Breast cancer remains a leading cause of cancer mortality worldwide, underscoring the urgent need for novel and effective therapeutic strategies. Eph receptor tyrosine kinases, particularly EphB4, exhibit diverse roles in cancer biology, acting as either tumor promoters or suppressors depending on the cellular environment and ligand engagement. EphB4 is frequently overexpressed in breast cancer and contributes to dysregulated signaling and tumor progression through the abnormal interaction with its ligand Ephrin-B2. We herein developed an improved anti-EphB4 monoclonal antibody, Eb4Mab-7-mG2a, which can be characterized as a subclass-switched IgG2a variant designed to enhance immune effector function, specifically antibody-dependent cellular cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC). Our findings showed that Eb4Mab-7-mG2a effectively blocked Ephrin-B2-induced ERK phosphorylation and proliferation in EphB4-positive MCF-7 breast cancer cells but had no effect on EphB4-knockout (KO) MCF-7 cells. Flow cytometry confirmed high-affinity binding between Eb4Mab-7-mG2a and EphB4-expressing cells, whereas in vitro assays demonstrated potent and selective ADCC and CDC activities against EphB4-positive tumor cells. In vivo experiments showed that Eb4Mab-7-mG2a significantly suppressed xenograft growth in models bearing EphB4-overexpressing CHO-K1 and EphB4-positive MCF-7, but showed no therapeutic effect in EphB4-negative CHO-K1 and EphB4-KO MCF-7 xenografts. Immunohistochemical analysis revealed reduced Ki-67 proliferation indices in treated tumors, supporting the antiproliferative effects of the developed antibody. Overall, these findings demonstrate that Eb4Mab-7-mG2a exerts dual-action antitumor activity through ligand blockade and immune effector engagement. Further evaluations in other EphB4-overexpressing cancers and in combination with immune checkpoint inhibitors are warranted. Humanization and tumor-selective engineering may enhance its clinical potential for precision oncology.

CD44 variants (CD44v) play essential roles in the promotion of tumor metastasis, maintenance of cancer stem cell properties, and resistance to treatments. Therefore, the development of anti-CD44v mAbs is essential for targeting CD44v-positive tumor cells. An anti-CD44v9 mAb, C44Mab-1 (mouse, IgG1, kappa), was previously established. C44Mab-1 recognizes the variant exon 9-encoded region and applies to multiple research techniques. A mouse IgG2a version of C44Mab-1 (C44Mab-1-mG2a) was generated to evaluate the in vitro and in vivo antitumor activities using gastric and colorectal cancer cell lines. C44Mab-1-mG2a showed a reactivity to CD44v3-10-overexpressed Chinese hamster ovary-K1 (CHO/CD44v3-10), gastric cancer MKN45, and colorectal cancer COLO205 in flow cytometry. C44Mab-1-mG2a exhibited both antibody-dependent cellular cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC) against CHO/CD44v3-10, MKN45, and COLO205. Furthermore, administration of C44Mab-1-mG2a significantly suppressed CHO/CD44v3-10, MKN45, and COLO205 xenograft tumor growth compared with control mouse IgG2a. These results indicated that C44Mab-1-mG2a, which possesses ADCC/CDC activities, could be applied to the mAb-based therapy against CD44v9-positive carcinomas.

Glypican 5 (GPC5) is a member of heparan sulfate proteoglycans and is anchored to the plasma membrane via glycosylphosphatidylinositol. GPC5 plays an essential role in kidney, limb, and brain development. Furthermore, GPC5 is expressed in some cancers, but whether it functions as a cancer-promoting or -suppressing factor remains unclear. Therefore, the development of versatile and specific anti-GPC5 monoclonal antibodies (mAbs) is desired to clarify the biological and pathological functions of GPC5. In this study, we successfully established an anti-human GPC5 mAb (clone G5Mab-1) using the Cell-Based Immunization and Screening method. G5Mab-1 is capable of using flow cytometric analysis. G5Mab-1 is specifically bound only to GPC5, not to the other GPC family members. The dissociation constant value of G5Mab-1 for GPC5-overexpressed Chinese hamster ovary K-1 (CHO/GPC5) cells was determined as 9.9 × 10-9 M. Furthermore, G5Mab-1 detected GPC5 in Western blot and immunohistochemistry using CHO/GPC5 cells. Therefore, the G5Mab-1 is highly versatile for basic research and is expected to contribute to clinical applications, such as antibody-based therapy and diagnosis of cancer.

Erythropoietin-producing hepatocellular (Eph) receptor A3 (EphA3) is a member of the Eph receptor family, which binds to its respective ligands, ephrins. These interactions are essential for normal development and tissue homeostasis. Dysregulation of EphA3 has been reported to be associated with human hematopoietic malignancies, making it a promising target for therapy and diagnosis. Due to the high similarity of the extracellular domain among Eph receptors (more than 33% amino acid identity), generating highly specific monoclonal antibodies (mAbs) is crucial. We developed anti-human EphA3 mAbs in this study using the Cell-Based Immunization and Screening (CBIS) method. Among them, the clone Ea3Mab-20 (IgG1, kappa) exhibited high affinity and specificity in flow cytometry. The dissociation constant values of Ea3Mab-20 for CHO/EphA3 and Jurkat cells were determined to be 9.0 ± 0.3 × 10-9 M and 1.4 ± 0.1 × 10-9 M, respectively. Ea3Mab-20 showed no cross-reactivity with other Eph receptors in flow cytometry. Furthermore, Ea3Mab-20 demonstrated the suitability for detecting formalin-fixed paraffin-embedded cell samples in immunohistochemistry. Therefore, Ea3Mab-20 is valuable mAb for basic research and is expected to contribute to the clinical application of mAb for cancer therapy and diagnosis.

Cadherins are key cell adhesion molecules that engage in extracellular homophilic binding. CDH15/M-cadherin is localized to the apical surface of muscle satellite cells (SCs), which play a critical role in tissue regeneration after injury. Although CDH15 is considered a marker of SCs, there is no anti-CDH15 monoclonal antibody (mAb) suitable for flow cytometry. We developed anti-CDH15 mAbs using the Cell-Based Immunization and Screening (CBIS) method containing a flow cytometry-based high-throughput screening. In flow cytometry, a clone Ca15Mab-1 (IgG1, κ) reacted with human CDH15-overexpressed Chinese hamster ovary-K1 (CHO/CDH15) cells. Furthermore, Ca15Mab-1 recognizes endogenous CDH15-expressing human osteosarcoma (Saos-2) and mouse myoblast (C2C12) cell lines. The dissociation constant values of Ca15Mab-1 for CHO/CDH15, Saos-2, and C2C12 were determined as 6.1 × 10-10 M, 7.9 × 10-10 M, and 9.8 × 10-10 M, respectively. Furthermore, Ca15Mab-1 can detect endogenous CDH15 in immunoblotting and immunohistochemistry. Ca15Mab-1, established by the CBIS method, is versatile for basic research and is expected to contribute to clinical studies such as antibody therapy.

Podoplanin (PDPN) is a highly glycosylated type I transmembrane protein. PDPN expression is observed in various normal tissues, including lymphatic endothelial cells, kidney podocytes, and type I alveolar epithelial cells in the lungs. Monoclonal antibodies (mAbs) targeting PDPN across different animal species have facilitated the identification of PDPN-positive cells. To date, we have developed anti-PDPN mAbs for over 20 species. These antibodies suit various applications, including flow cytometry, immunoblotting, and immunohistochemistry. In this study, we generated an anti-hippopotamus PDPN (hipPDPN) mAb, PMab-322 (mouse IgG2a, kappa), using the Cell-Based Immunization and Screening (CBIS) method. PMab-322 exhibited strong reactivity to hipPDPN-overexpressed Chinese hamster ovary-K1 and demonstrated moderate affinity (K D: 4.4 × 10-8 M) in a flow cytometry-based measurement. PMab-322 specifically recognizes hipPDPN but does not cross-react with PDPN from 23 other species. Furthermore, PMab-322 successfully detected hipPDPN in both immunoblotting and immunohistochemistry. These findings highlight the potential of PMab-322 for pathological analyses of hippopotamus-derived tissues.

Eph receptor B2 (EphB2) overexpression is associated with poor clinical outcomes in various tumors. EphB2 is involved in malignant tumor progression through the promotion of invasiveness and metastasis. Genetic and transcriptome analyses implicated that EphB2 is a therapeutic target for specific tumor types. A monoclonal antibody (mAb) is one of the essential therapeutic strategies for EphB2-positive tumors. We previously developed an anti-EphB2 mAb, Eb2Mab-12 (IgG1, kappa), by immunizing mice with EphB2-overexpressed glioblastoma. Eb2Mab-12 specifically reacted with the EphB2-overexpressed Chinese hamster ovary-K1 (CHO/EphB2) and some cancer cell lines in flow cytometry. In this study, we engineered Eb2Mab-12 into a mouse IgG2a type (Eb2Mab-12-mG2a) and a human IgG1-type (Eb2Mab-12-hG1) mAb. Eb2Mab-12-mG2a and Eb2Mab-12-hG1 retained the reactivity to EphB2-positive cells and exerted antibody-dependent cellular cytotoxicity and complement-dependent cytotoxicity in the presence of effector cells and complements, respectively. In CHO/EphB2, triple-negative breast cancer, and lung mesothelioma xenograft models, both Eb2Mab-12-mG2a and Eb2Mab-12-hG1 exhibited potent antitumor efficacy. These results indicated that Eb2Mab-12-derived mAbs could be applied to mAb-based therapy against EphB2-positive tumors.

Background: Podocalyxin (PODXL) has been identified as a promising therapeutic target and a potential diagnostic biomarker in various tumors. Despite the therapeutic potential of anti-PODXL monoclonal antibodies (mAbs), their further development has been limited by concerns regarding potential on-target off-tumor toxicities. To minimize adverse effects on normal tissues, developing a cancer-specific mAb (CasMab) against PODXL is essential. Methods: Our group established a cancer-specific anti-PODXL mAb, PcMab-60 (IgM, κ), through the screening of over one hundred hybridoma clones. In this study, PcMab-60 was engineered into a humanized IgG1-type mAb (humPcMab-60), and its antitumor activity was examined using mouse xenograft models of pancreatic ductal adenocarcinoma (PDAC) and colorectal cancer. Results: HumPcMab-60 retains cancer-specific reactivity; humPcMab-60 reacted to PDAC cell lines (PK-45H and MIA PaCa-2) and the colorectal cancer cell line (Caco-2), but not to a normal lymphatic endothelial cell line in flow cytometry. Furthermore, humPcMab-60 exerted antibody-dependent cellular cytotoxicity and complement-dependent cytotoxicity against PODXL-expressing cell lines and showed antitumor effects against the tumor xenografts. Conclusions: A humanized anti-PODXL CasMab, humPcMab-60, could be a promising mAb-based tumor therapy.

The chemokine receptors possess seven transmembrane helices connected by an extracellular N‐terminal region, three extracellular loops (ECL1–3), three intracellular loops, and an intracellular C‐terminal region. Specific monoclonal antibodies (mAbs) against chemokine receptors for flow cytometry have been developed using Cell-Based Immunization and Screening, and the N-terminal peptide immunization methods. However, there are few reports on the establishment of anti-chemokine receptor mAbs through immunization with ECL peptides. Here, an anti-mouse C–C chemokine receptor type 7 (mCCR7) mAb, C7Mab-2 (rat immunoglobulin G2b, kappa), was established through immunization with the ECL3 peptide. C7Mab-2 demonstrated reactivity to mCCR7-overexpressed Chinese hamster ovary-K1 (CHO/mCCR7) cells in flow cytometry, which was inhibited by the ECL3 peptide. C7Mab-2 did not show cross-reactivity with other mouse CC, CXC, CX3C, and XC chemokine receptors. The dissociation constant value of C7Mab-2 was determined to be 2.8 × 10−9 M for CHO/mCCR7 cells. Furthermore, C7Mab-2 detected mCCR7 in immunohistochemistry. This strategy could accelerate the development of novel chemokine receptor mAbs with high affinity and specificity.

Erythropoietin-producing hepatocellular receptor A8 (EphA8) is a type I transmembrane protein that belongs to the largest erythropoietin-producing hepatocellular (Eph) family among receptor tyrosine kinases. By binding to its membrane-bound ephrin-A or ephrin-B ligands on adjacent cells, Eph receptors form complexes and mediate bidirectional signaling activities, triggering cell-cell adhesion and repulsion. Increased expression of EphA8 correlates with poor prognosis in some types of cancer. Therefore, developing sensitive monoclonal antibodies (mAbs) for EphA8 has been desired for treatment, diagnosis, and further basic research. In particular, there are no anti-EphA8 mAbs that can be used for flow cytometry. A novel, specific, and sensitive anti-human EphA8 mAb, which applies to flow cytometry, clone Ea8Mab-9 (mouse immunoglobulin G1, kappa), was established using the Cell-Based Immunization and Screening method. Ea8Mab-9 reacted with EphA8-overexpressed Chinese hamster ovary-K1 cells (CHO/EphA8) and EphA8-overexpressed LN229 glioblastoma cells (LN229/EphA8) in flow cytometry. Notably, Ea8Mab-9 did not recognize other members of the Eph receptor family. Furthermore, Ea8Mab-9 demonstrated a high binding affinity for CHO/EphA8 and LN229/EphA8, with dissociation constants of 1.3 × 10-9 M and 1.6 × 10-9 M, respectively. The reaction of Ea8Mab-9 with CHO/EphA8 was completely blocked by a recombinant EphA8 protein. Ea8Mab-9 could be useful for analyzing the EphA8-related biological responses using flow cytometry, owing to its high affinity and specificity.

Erythropoietin-producing hepatocellular receptor A1 (EphA1) is one of the Eph receptor family members, the largest group of receptor tyrosine kinases. EphA1 is expressed in various tissues and regulates cellular homeostasis by interacting with its membrane-bound ephrin ligands and other receptors. EphA1 critically correlates with the pathogenesis in several disorders, including Alzheimer's disease and cancers. Therefore, establishing sensitive monoclonal antibodies (mAbs) for EphA1 has been desired for basic research, diagnosis, and treatment. In this study, a novel specific and sensitive anti-human EphA1 mAb, clone Ea1Mab-30 (mouse IgG1, kappa), was established by the Cell-Based Immunization and Screening (CBIS) method. Ea1Mab-30 demonstrated reactivity with an EphA1-overexpressed Chinese hamster ovary-K1 cell line (CHO/EphA1), an endogenously EphA1-expressing bladder carcinoma cell line (5637), and a colorectal adenocarcinoma cell line (Caco-2) in flow cytometry. Crossreactivities of Ea1Mab-30 with other Eph receptors were not observed. Furthermore, the values of apparent binding affinity for CHO/EphA1 and 5637 were determined to be 8.9 × 10-9 M and 1.7 × 10-9 M, respectively. Furthermore, Ea1Mab-30 detected EphA1 protein in CHO/EphA1 and 5637 lysates using Western blot analysis. Ea1Mab-30 also clearly stained EphA1 of formalin-fixed paraffin-embedded CHO/EphA1 using immunohistochemistry. Ea1Mab-30, established by CBIS method, could help analyze the EphA1-contributed cellular functions and have potential applications in pathological diagnosis and treatment with specificity and high affinity for EphA1-expressing cells.

CXC chemokine receptor 1 (CXCR1) is an important regulator for neutrophil granulocyte activation through binding to the ligand interleukin-8 (IL-8). Upon binding to IL-8, CXCR1 activates downstream signaling, critical for innate and adaptive immune responses. The IL-8-CXCR1 axis also plays an important role in tumor progression, especially in the tumor microenvironment. CXCR1 antagonists or anti-IL-8 monoclonal antibodies (mAbs) have been developed and evaluated in clinical trials for inflammatory diseases and tumors. In this study, we developed novel mAbs for mouse CXCR1 (mCXCR1) using the N-terminal peptide immunization. Among the established anti-mCXCR1 mAbs, Cx1Mab-8 (rat IgG2b, kappa) recognized mCXCR1-overexpressed Chinese hamster ovary-K1 (CHO/mCXCR1) and mCXCR1-overexpressed LN229 (LN229/mCXCR1) by flow cytometry. The dissociation constant (K D) values of Cx1Mab-8 for CHO/mCXCR1 and LN229/mCXCR1 were determined as 4.1 × 10-10 M and 1.5 × 10-9 M, respectively. These results indicated that Cx1Mab-8 is useful for detecting mCXCR1 by flow cytometry with high affinity and could contribute to obtaining the proof of concept in preclinical studies.

Podoplanin (PDPN), also referred to as T1α/Aggrus, is a type I transmembrane sialoglycoprotein that plays a crucial role in invasiveness, stemness, and epithelial-to-mesenchymal transition, all of which contribute to the malignant progression of tumors. Therefore, a monoclonal antibody (mAb) against PDPN has been evaluated in preclinical models as a promising tumor therapy strategy. However, PDPN plays an essential role in normal development, such as in the development of the lungs. On-target toxicity by anti-PDPN mAbs to normal cells should be avoided to minimize adverse effects. A cancer-specific mAb against PDPN, PMab-117 (rat IgM, kappa), was previously established. This study engineered the humanized IgG1 version (humPMab-117) to investigate antitumor activity. Flow cytometry analysis confirmed that humPMab-117 recognized PDPN-overexpressed glioma LN229 (LN229/PDPN) cells as well as PDPN-positive PC-10 (human lung squamous cell carcinoma) and LN319 (human glioblastoma) cells. In contrast, humPMab-117 did not react with normal epithelial cells from the lung bronchus, gingiva, mammary gland, corneal, and normal kidney podocytes, suggesting that humPMab-117 retains cancer-specific reactivity. Furthermore, humPMab-117 effectively induced antibody-dependent cellular cytotoxicity and complement-dependent cytotoxicity against LN229/PDPN, PC-10, and LN319 cells. In the xenograft tumor models, humPMab-117 demonstrated strong antitumor efficacy. These results suggest the potential of humPMab-117 as a therapeutic antibody for treating PDPN-positive malignant tumors.

C-C chemokine receptor type 7 (CCR7) is a member of the G protein-coupled receptor family and functions as a lymph node-homing receptor for immune cells. Upon ligand binding, CCR7 promotes the migration of immune cells to secondary lymphoid organs. In cancers, CCR7 has been revealed as a critical molecule in lymph node metastasis. Consequently, anti-CCR7 monoclonal antibodies (mAbs) have been developed as cancer therapeutic agents. In this study, we established an anti-mouse CCR7 (mCCR7) mAb, C7Mab-7 (rat IgG1, kappa) using the Cell-Based Immunization and Screening (CBIS) method. C7Mab-7 demonstrated high sensitivity in flow cytometry. The dissociation constant (K D) value of C7Mab-7 was determined to be 2.5 × 10⁻⁹ M for mCCR7-overexpressed Chinese hamster ovary-K1 (CHO/mCCR7) cells. Furthermore, C7Mab-7 detected mCCR7 with high sensitivity in western blot and immunohistochemistry. C7Mab-7, developed by the CBIS method, accelerates the development of CCR7-targeted antibody therapies and cancer diagnostics.

Erythropoietin-producing hepatocellular receptor B6 (EphB6) is a member of the largest Eph subfamily of receptor tyrosine kinases. EphB6 is widely expressed in various tissues and regulates cellular homeostasis by interacting with its membrane-bound ephrin ligands and other receptors. EphB6 is involved in cancer pathology despite lacking kinase activity. Developing sensitive monoclonal antibodies (mAbs) for EphB6 has been desired for treatment, diagnosis, and further analysis of EphB6. This study established a novel specific and sensitive anti-human EphB6 mAb clone Eb6Mab-3 (mouse IgG1, kappa) by the Cell-Based Immunization and Screening (CBIS) method. In flow cytometry, Eb6Mab-3 demonstrated reactivity with EphB6-overexpressed Chinese hamster ovary-K1 cells (CHO/EphB6) and endogenously EphB6-expressing DLD-1 colorectal cancer cells. Cross-reactivity of Eb6Mab-3 was not observed. Eb6Mab-3 demonstrated a moderate binding affinity (dissociation constant; K D) for CHO/EphB6 (K D: 2.6 ± 1.0 × 10-8 M) and a high binding affinity for DLD-1 (K D: 3.4 ± 1.3 × 10-9 M). Eb6Mab-3 can detect EphB6 protein in CHO/EphB6 lysate in Western blot. Eb6Mab-3, established by the CBIS method, could be valuable for analyzing the EphB6-associated cellular functions and has potential applications in diagnosis and treatment with specificity and high affinity for cancer cells.

Malignant neoplasms arise within a region of chronic inflammation caused by tissue injuries. Inflammation is a key factor involved in all aspects of tumorigenesis including initiation, proliferation, invasion, angiogenesis, and metastasis. Interleukin-1 (IL-1) plays critical functions in tumor development with influencing the tumor microenvironment and promoting cancer progression. However, the mechanism of continuous activation of IL-1-mediated inflammatory pathway in tumor has not been fully elucidated. This study provides a novel mechanism of the autocrine activation of IL-1 signaling in squamous cell carcinoma (SCC) through a novel oncoprotein, TSC-22 homologous gene-1 (THG-1, also known as TSD22D4). The RNA sequencing analysis revealed that THG-1 overexpression enhances the transcription of NF-κB targets including IL1A, IL1B, TNFA, and IL8. Furthermore, THG-1 knockdown reduced the responsiveness to IL-1 through suppression of NF-κB nuclear translocation. To elucidate the mechanism, we focused on a THG-1 interacting protein, NRBP1. We found that NRBP1 facilitates the degradation of TRAF6 through its E3 ubiquitin ligase activity. THG-1 bound to NRBP1 and suppressed the degradation of TRAF6. Furthermore, THG-1 knockdown reduced TRAF6 abundance and NF-κB activity in SCC cells. Public database analyses of head and neck SCC revealed that high expression of THG-1 is associated with activation of the IL-1 and TNF pathways, which share TRAF6 in the signal transductions. Finally, THG-1 abundance in laryngeal SCC specimens is elevated in patients with recurrence. These results indicated that THG-1 drives the self-sufficiency of IL-1-mediated inflammatory pathway, which could contribute to the future diagnosis and immune therapy of SCCs. Implications: An oncoprotein THG-1/TSD22D4 activates the IL-1-mediated inflammatory pathway through suppression of TRAF6 degradation, which mediates the continuous inflammation in tumors.

Monoclonal antibody (mAb) and cell-based immunotherapies represent cutting-edge strategies for cancer treatment. However, safety concerns persist due to the potential targeting of normal cells that express reactive antigens. Therefore, it is crucial to develop cancer-specific mAbs (CasMabs) that can bind to cancer-specific antigens and exhibit antitumor activity in vivo, thereby reducing the risk of adverse effects. We previously screened mAbs targeting human epidermal growth factor receptor 2 (HER2) and successfully developed a cancer-specific anti-HER2 mAb, H2Mab-250/H2CasMab-2 (mouse IgG1, kappa). In this study, we assessed both the in vitro and in vivo antitumor efficacy of the humanized H2Mab-250 (humH2Mab-250). Although humH2Mab-250 showed lower reactivity to HER2-overexpressed Chinese hamster ovary-K1 (CHO/HER2) and breast cancer cell lines (BT-474 and SK-BR-3) than trastuzumab in flow cytometry, both humH2Mab-250 and trastuzumab showed similar antibody-dependent cellular cytotoxicity (ADCC) against CHO/HER2 and the breast cancer cell lines in the presence of effector splenocytes. In addition, humH2Mab-250 exhibited significant complement-dependent cellular cytotoxicity (CDC) in CHO/HER2 and the breast cancer cell lines compared to trastuzumab. Furthermore, humH2Mab-250 possesses compatible in vivo antitumor effects against CHO/HER2 and breast cancer xenografts with trastuzumab. These findings highlight the distinct roles of ADCC and CDC in the antitumor effects of humH2Mab-250 and trastuzumab and suggest a potential direction for the clinical development of humH2Mab-250 for HER2-positive tumors.

Ephrin type-B receptor 2 (EphB2) is a member of the Eph family tyrosine kinase receptors. EphB2 binds to ephrin-B1, ephrin-B2, and ephrin-B3, which are critical regulators of vascular and neural development, influencing cell migration and axon guidance. EphB2 is overexpressed in several tumors, including glioma, breast cancer, hepatocellular carcinoma, and malignant mesothelioma, where it functions as a tumor promoter. Therefore, the development of monoclonal antibodies (mAbs) targeting EphB2 is essential for the diagnosis and treatment of EphB2-positive tumors. In this study, we developed novel mAbs for human EphB2 using the Cell-Based Immunization and Screening method. Among the established anti-EphB2 mAbs, Eb2Mab-12 (mouse IgG1, kappa) showed reactivity toward EphB2-overexpressed Chinese hamster ovary-K1 cells (CHO/EphB2) and an endogenously EphB2-expressing cancer cell line (LS174T), as confirmed by flow cytometry. The dissociation constant (KD) values of Eb2Mab-12 for CHO/EphB2 and LS174T were determined to be 1.7 × 10−9 M and 4.4 × 10−10 M, respectively, using flow cytometry. Furthermore, Eb2Mab-12 exhibited no cross-reactivity with other members of the EphA and EphB receptors. These results indicate that Eb2Mab-12 possesses high affinity and specificity in detecting EphB2, suggesting its potential application in tumor therapy.

The CXC chemokine receptor 5 (CXCR5) is a member of the G protein-coupled receptor family that is highly expressed in B cells and a subset of T cells, such as T follicular helper cells. Various types of cancers, including non-small cell lung cancer, breast cancer, and prostate cancer, also express CXCR5. Therefore, antibodies that specifically bind to CXCR5 could be useful for clarification of the mechanisms of cancer progression. In this study, we aimed to develop high-affinity monoclonal antibodies targeting mouse CXCR5 (mCXCR5) for flow cytometry. The established anti-mCXCR5 mAb (Cx5Mab-3; rat IgG2b, kappa), demonstrated reactivity with mCXCR5-overexpressed Chinese hamster ovary (CHO)-K1 (CHO/mCXCR5) in flow cytometry. Kinetic analyses using flow cytometry indicated that the dissociation constants (KD) of Cx5Mab-3 for CHO/mCXCR5 cell is 7.2 × 10−10 M. Furthermore, Cx5Mab-3 did not cross-react with other mouse CC, CXC, CX3C, and XC chemokine receptors. These results indicate that Cx5Mab-3 is useful for detecting mCXCR5 in flow cytometry with high affinity and specificity.

C-C motif chemokine receptor-8 (CCR8) belongs to class A of G protein-coupled receptors. CCR8 interacts with the specific chemokine ligand CCL1/I-309 in humans, which is produced by various cells, including tumor-associated macrophages and regulatory T cells (Treg). CCR8 is highly expressed on Treg and T-helper 2 cells recruited to the inflammation site and is implicated in allergy, asthma, and cancer progression. CCR8+Treg cells have been suggested an important regulator in the immunosuppressive tumor microenvironment. Therefore, it has been proposed for use in the development of sensitive monoclonal antibodies targeting CCR8. This study developed a specific mAb for human CCR8 (hCCR8), which is useful for flow cytometry by employing the Cell-Based Immunization and Screening (CBIS) method. The established anti-hCCR8 mAb (C8Mab-21; mouse IgM, kappa) demonstrated reactivity with hCCR8-overexpressed Chinese hamster ovary-K1 (CHO/hCCR8) cells, TALL-1 (human adult acute T-lymphoblastic leukemia), CCRF-HSB2 (human T-lymphoblastic leukemia), and natural killer cells expressing endogenous hCCR8, as confirmed by flow cytometry. Furthermore, EC50 values of C8Mab-21 for CHO/hCCR8 and TALL-1 were determined as 6.5 × 10−8 M and 2.0 × 10−8 M, respectively. C8Mab-21, established by the CBIS method, provides a useful tool for analyzing the hCCR8-related biological response using flow cytometry.

Leukocyte migration is an essential function of innate and adaptive immune responses. Chemokines and their receptors control the migration system. The abundance of chemokines is controlled by atypical chemokine receptors (ACKRs), chemokine receptor-like molecules that do not couple to the G protein signaling pathways. Among them, ACKR4 regulates dendritic cell migration by controlling the ligands and is involved in tumor development in mouse models. Because no anti-mouse ACKR4 (mACKR4) monoclonal antibody (mAb) for flow cytometry has been reported, this study aimed to develop a novel mAb for mACKR4. Among the established anti-mACKR4 mAbs, A4Mab-1 (rat IgG2b, kappa), A4Mab-2 (rat IgG2b, kappa), and A4Mab-3 (rat IgG2b, kappa) recognized mACKR4-overexpressed Chinese hamster ovary-K1 (CHO/mACKR4) by flow cytometry. The dissociation constant (K D) values of A4Mab-1, A4Mab-2, and A4Mab-3 for CHO/mACKR4 were determined as 6.0 × 10-9 M, 1.3 × 10-8 M, and 1.7 × 10-9 M, respectively. Furthermore, A4Mab-1 and A4Mab-2 could detect mACKR4 by western blotting. These results indicated that A4Mab-1, A4Mab-2, and A4Mab-3 help to detect mACKR4 by flow cytometry and western blotting and obtain the proof of concept in preclinical models.

Podoplanin (PDPN) overexpression is associated with poor clinical outcomes in various tumors. PDPN is involved in malignant tumor progression by promoting invasiveness and metastasis. Therefore, PDPN is considered a promising target of monoclonal antibody (mAb)-based therapy. Because PDPN also plays an essential role in normal cells such as kidney podocytes, cancer specificity is required to reduce adverse effects on normal cells. We developed a cancer-specific mAb (CasMab) against PDPN, PMab-117 (rat IgM, kappa), by immunizing rats with PDPN-overexpressed glioblastoma cells. The recombinant mouse IgG2a-type PMab-117 (PMab-117-mG2a) reacted with the PDPN-positive tumor PC-10 and LN319 cells but not with PDPN-knockout LN319 cells in flow cytometry. PMab-117-mG2a did not react with normal kidney podocytes and normal epithelial cells from the lung bronchus, mammary gland, and corneal. In contrast, one of the non-CasMabs against PDPN, NZ-1, showed high reactivity to PDPN in both tumor and normal cells. Moreover, PMab-117-mG2a exerted antibody-dependent cellular cytotoxicity in the presence of effector splenocytes. In the human tumor xenograft models, PMab-117-mG2a exhibited potent antitumor effects. These results indicated that PMab-117-mG2a could be applied to antibody-based therapy against PDPN-expressing human tumors while reducing the adverse effects.

CD44 regulates cell adhesion, proliferation, survival, and stemness and has been considered a tumor therapy target. CD44 possesses the shortest CD44 standard (CD44s) and a variety of CD44 variant (CD44v) isoforms. Since the expression of CD44v is restricted in epithelial cells and carcinomas compared to CD44s, CD44v has been considered a promising target for monoclonal antibody (mAb) therapy. We previously developed an anti-CD44v10 mAb, C44Mab-18 (IgM, kappa), to recognize the variant exon 10-encoded region. In the present study, a mouse IgG2a version of C44Mab-18 (C44Mab-18-mG2a) was generated to evaluate the antitumor activities against CD44-positive cells compared with the previously established anti-pan CD44 mAb, C44Mab-46-mG2a. C44Mab-18-mG2a exhibited higher reactivity compared with C44Mab-46-mG2a to CD44v3–10-overexpressed CHO-K1 (CHO/CD44v3–10) and oral squamous cell carcinoma cell lines (HSC-2 and SAS) in flow cytometry. C44Mab-18-mG2a exerted a superior antibody-dependent cellular cytotoxicity (ADCC) against CHO/CD44v3–10. In contrast, C44Mab-46-mG2a showed a superior complement-dependent cytotoxicity (CDC) against CHO/CD44v3–10. A similar tendency was observed in ADCC and CDC against HSC-2 and SAS. Furthermore, administering C44Mab-18-mG2a or C44Mab-46-mG2a significantly suppressed CHO/CD44v3–10, HSC-2, and SAS xenograft tumor growth compared with the control mouse IgG2a. These results indicate that C44Mab-18-mG2a could be a promising therapeutic regimen for CD44v10-positive tumors.

Cancer-specific monoclonal antibodies (CasMabs) that recognize cancer-specific antigens with in vivo antitumor efficacy are innovative therapeutic strategies for minimizing adverse effects. We previously established a cancer-specific anti-human epidermal growth factor receptor 2 (HER2) monoclonal antibody (mAb), H2Mab-250/H2CasMab-2. In flow cytometry and immunohistochemistry, H2Mab-250 reacted with HER2-positive breast cancer cells but did not show reactivity to normal epithelial cells. In contrast, a clinically approved anti-HER2 mAb, trastuzumab, strongly recognizes both breast cancer and normal epithelial cells in flow cytometry. The human IgG1 version of H2Mab-250 (H2Mab-250-hG1) possesses compatible in vivo antitumor effects against breast cancer xenografts to trastuzumab despite the lower affinity and effector activation than trastuzumab in vitro. This study compared the antibody-dependent cellular cytotoxicity (ADCC) and complement-dependent cellular cytotoxicity (CDC) between H2Mab-250-hG1 and trastuzumab. Both H2Mab-250-hG1 and trastuzumab showed ADCC activity against HER2-overexpressed Chinese hamster ovary -K1 and breast cancer cell lines (BT-474 and SK-BR-3) in the presence of human natural killer cells. Some tendency was observed where trastuzumab showed a more significant ADCC effect compared to H2Mab-250-hG1. Importantly, H2Mab-250-hG1 exhibited superior CDC activity in these cells compared to trastuzumab. Similar results were obtained in the mouse IgG2a types of both H2Mab-250 and trastuzumab. These results suggest the different contributions of ADCC and CDC activities to the antitumor effects of H2Mab-250-hG1 and trastuzumab, and indicate a future direction for the clinical development of H2Mab-250-hG1 against HER2-positive tumors.

One of the G protein-coupled receptors, C-C chemokine receptor 5 (CCR5), is an important regulator for the activation of T and B lymphocytes, dendritic cells, natural killer cells, and macrophages. Upon binding to its ligands, CCR5 activates downstream signaling, which is an important regulator in the innate and adaptive immune response through the promotion of lymphocyte migration and the secretion of proinflammatory cytokines. Anti-CCR5 monoclonal antibodies (mAbs) have been developed and evaluated in clinical trials for tumors and inflammatory diseases. In this study, we developed novel mAbs for mouse CCR5 (mCCR5) using the N-terminal peptide immunization. Among the established anti-mCCR5 mAbs, C5Mab-4 (rat IgG2a, kappa) and C5Mab-8 (rat IgG1, kappa), recognized mCCR5-overexpressing Chinese hamster ovary-K1 (CHO/mCCR5) and an endogenously mCCR5-expressing cell line (L1210) by flow cytometry. The dissociation constant (KD) values of C5Mab-4 and C5Mab-8 for CHO/mCCR5 were determined as 3.5 × 10-8 M and 7.3 × 10-9 M, respectively. Furthermore, both C5Mab-4 and C5Mab-8 could detect mCCR5 by western blotting. These results indicated that C5Mab-4 and C5Mab-8 are useful for detecting mCCR5 by flow cytometry and western blotting and provide a possibility to obtain the proof of concept in preclinical studies.

C-C chemokine receptor 5 (CCR5), a member of the G protein-coupled receptor family, is the most common coreceptor for the human immunodeficiency virus type 1. CCR5 is also involved in the pathogenesis of tumors and inflammatory diseases. The CCR5 antagonists including monoclonal antibodies (mAbs) have been developed and evaluated in clinical trials. In this study, we developed novel mAbs for mouse CCR5 (mCCR5) using the Cell-Based Immunization and Screening (CBIS) method. One of the established anti-mCCR5 mAbs, C5Mab-2 (rat IgG2b, kappa), reacted with mCCR5-overexpressed Chinese hamster ovary-K1 (CHO/mCCR5) and an endogenously mCCR5-expressing cell line (L1210) by flow cytometry. Using flow cytometry, the dissociation constant (KD) of C5Mab-2 for CHO/mCCR5 was determined as 4.3 × 10-8 M. These results indicated that C5Mab-2 is useful for the detection of mCCR5 in flow cytometry and may be applicable to obtain the proof of concept in preclinical studies.

The C-X-C motif chemokine receptor-1 (CXCR1) is a rhodopsin-like G-protein-coupled receptor, expressed on the cell surface of immune cells and tumors. CXCR1 interacts with some C-X-C chemokines, such as CXCL6, CXCL7, and CXCL8/interleukin-8, which are produced by various cells. Since CXCR1 is involved in several diseases including tumors and diabetes mellitus, drugs targeting CXCR1 have been developed. Therefore, the development of sensitive monoclonal antibodies (mAbs) for CXCR1 has been desired for the diagnosis and treatment. This study established a novel anti-mouse CXCR1 (mCXCR1) mAb, Cx1Mab-1 (rat IgG1, kappa), using the Cell-Based Immunization and Screening method. Cx1Mab-1 reacted with mCXCR1-overexpressed Chinese hamster ovary-K1 (CHO/mCXCR1) and mCXCR1-overexpressed LN229 glioblastoma (LN229/mCXCR1) in flow cytometry. Cx1Mab-1 demonstrated a high binding affinity for CHO/mCXCR1 and LN229/mCXCR1 with a dissociation constant of 2.6 × 10-9 M and 2.1 × 10-8 M, respectively. Furthermore, Cx1Mab-1 could detect mCXCR1 by Western blot analysis. These results indicated that Cx1Mab-1 is useful for detecting mCXCR1, and provides a possibility for targeting mCXCR1-expressing cells in vivo experiments.

The giant panda (Ailuropoda melanoleuca) is one of the important species in worldwide animal conservation. Because it is essential to understand the disease of giant panda for conservation, histopathological analyses of tissues are important to understand the pathogenesis. However, monoclonal antibodies (mAbs) against giant panda-derived proteins are limited. Podoplanin (PDPN) is an essential marker of lung type I alveolar epithelial cells, kidney podocytes, and lymphatic endothelial cells. PDPN is also overexpressed in various human tumors, which are associated with poor prognosis. Here, an anti-giant panda PDPN (gpPDPN) mAb, PMab-314 (mouse IgG1, kappa) was established using the Cell-Based Immunization and Screening method. PMab-314 recognized N-terminal PA16-tagged gpPDPN-overexpressed Chinese hamster ovary-K1 cells (CHO/PA16-gpPDPN) in flow cytometry. The KD value of PMab-314 for CHO/PA16-gpPDPN was determined as 1.3 × 10-8 M. Furthermore, PMab-314 is useful for detecting gpPDPN in western blot analysis. These findings indicate that PMab-314 is a useful tool for the analyses of gpPDPN-expressed cells.

C-X-C motif chemokine receptor 3 (CXCR3, CD183) is a G-protein-coupled receptor for CXCL9, CXCL10, and CXCL11. CXCR3 induces chemotaxis of immune cells and promotes inflammation. Various mouse models have been developed to mimic the pathogenesis of diseases and used in the evaluation of therapeutics for these diseases. Although CXCR3 is an attractive target to suppress inflammation, anti-CXCR3 therapeutic agents have not been approved. In this study, we established a novel anti-mouse CXCR3 (mCXCR3) monoclonal antibody, Cx3Mab-4 (rat IgG1, kappa), using the Cell-Based Immunization and Screening method. Flow cytometric analysis demonstrated that Cx3Mab-4 bound to mCXCR3-overexpressed Chinese hamster ovary-K1 (CHO/mCXCR3) cells, but did not react to parental CHO-K1 cells. The dissociation constant of Cx3Mab-4 was determined as 1.3 × 10-9 M, indicating that Cx3Mab-4 possesses a high affinity to mCXCR3-expressing cells. Cx3Mab-4 could be useful for targeting CXCR3-expressing cells in preclinical mouse models.

A cell-surface ectonucleotidase CD39 mediates the conversion of extracellular adenosine triphosphate into immunosuppressive adenosine with another ectonucleotidase CD73. The elevated adenosine in the tumor microenvironment attenuates antitumor immunity, which promotes tumor cell immunologic escape and progression. Anti-CD39 monoclonal antibodies (mAbs), which suppress the enzymatic activity, can be applied to antitumor therapy. Therefore, an understanding of the relationship between the inhibitory activity and epitope of mAbs is important. We previously established an anti-mouse CD39 (anti-mCD39) mAb, C39Mab-1 using the Cell-Based Immunization and Screening method. In this study, we determined the critical epitope of C39Mab-1 using flow cytometry. We performed the PA tag (12 amino acids [aa])-substituted analysis (named PA scanning) and RIEDL tag (5 aa)-substituted analysis (named RIEDL scanning) to determine the critical epitope of C39Mab-1 using flow cytometry. By the combination of PA scanning and RIEDL scanning, we identified the conformational epitope, spanning three segments of 275-279, 282-291, and 306-323 aa of mCD39. These analyses would contribute to the identification of the conformational epitope of membrane proteins.

Bladder cancer is one of the most common cancers among men worldwide. Although multiple genomic mutations and epigenetic alterations have been identified, an efficacious molecularly targeted therapy has yet to be established. Therefore, a novel approach is anticipated. Glycoprotein nonmetastatic melanoma protein B (GPNMB) is a type I transmembrane glycoprotein that is highly expressed in various cancers. In this study, we evaluated bladder cancer patient samples and found that GPNMB protein abundance is associated with high-grade tumors, and both univariate and multivariate analyses showed that GPNMB is a prognostic factor. Furthermore, the prognosis of patients with high GPNMB levels was significantly poorer in those with nonmuscle invasive bladder cancer (NMIBC) than in those with muscle invasive bladder cancer (MIBC). We then demonstrated that knockdown of GPNMB in MIBC cell lines with high GPNMB inhibits cellular migration and invasion, whereas overexpression of GPNMB further enhances cellular migration and invasion in MIBC cell lines with originally low GPNMB. Therefore, we propose that GPNMB is one of multiple driver molecules in the acquisition of cellular migratory and invasive potential in bladder cancers. Moreover, we revealed that the tyrosine residue in the hemi-immunoreceptor tyrosine-based activation motif (hemITAM) is required for GPNMB-induced cellular motility.

Monoclonal antibody (mAb)-based and/or cell-based immunotherapies provide innovative approaches to cancer treatments. However, safety concerns over targeting normal cells expressing reactive antigens still exist. Therefore, the development of cancer-specific mAbs (CasMabs) that recognize cancer-specific antigens with in vivo antitumor efficacy is required to minimize the adverse effects. We previously screened anti-human epidermal growth factor receptor 2 (HER2) mAbs and successfully established a cancer-specific anti-HER2 mAb, H2Mab-250/H2CasMab-2 (IgG1, kappa). In this study, we showed that H2Mab-250 reacted with HER2-positive breast cancer cells but did not show reactivity to normal epithelial cells in flow cytometry. In contrast, a clinically approved anti-HER2 mAb, trastuzumab, recognized both breast cancer and normal epithelial cells. We further compared the affinity, effector activation, and antitumor effect of H2Mab-250 with trastuzumab. The results showed that H2Mab-250 exerted a comparable antitumor effect with trastuzumab in the mouse xenograft models of BT-474 and SK-BR-3, although H2Mab-250 possessed a lower affinity and effector activation than trastuzumab in vitro. H2Mab-250 could contribute to the development of chimeric antigen receptor-T or antibody-drug conjugates without adverse effects for breast cancer therapy.

CD39 is involved in adenosine metabolism by converting extracellular ATP to adenosine. As extracellular adenosine plays a critical role in the immune suppression of the tumor microenvironment, the inhibition of CD39 activity by monoclonal antibodies (mAbs) is one of the important strategies for tumor therapy. This study developed specific and sensitive mAbs for mouse CD39 (mCD39) using the Cell-Based Immunization and Screening method. The established anti-mCD39 mAb, C39Mab-1 (rat IgG2a, kappa), reacted with mCD39-overexpressed Chinese hamster ovary-K1 (CHO/mCD39) by flow cytometry. The kinetic analysis using flow cytometry indicated that the dissociation constant of C39Mab-1 for CHO/mCD39 was 7.3 × 10-9 M. Furthermore, C39Mab-1 detected the lysate of CHO/mCD39 by western blot analysis. These results indicated that C39Mab-1 is useful for the detection of mCD39 in many functional studies.

Immune checkpoint blockade therapy has shown successful clinical outcomes in multiple human cancers. In dogs, several types of tumors resemble human tumors in many respects. Therefore, several groups have developed the anti-dog programmed cell death ligand 1 (dPD-L1) monoclonal antibodies (mAbs) and showed efficacy in several canine tumors. To examine the abundance of dPD-L1 in canine tumors, anti-dPD-L1 diagnostic mAbs for immunohistochemistry are required. In this study, we immunized the peptide in the dPD-L1 intracellular domain, and established anti-dPD-L1 mAbs, L1Mab-352 (mouse IgG1, kappa), and L1Mab-354 (mouse IgG1, kappa). In enzyme-linked immunosorbent assay, L1Mab-352 and L1Mab-354 showed high-binding affinity to the dPD-L1 peptide, and the dissociation constants (KD) were determined as 6.9 × 10-10 M and 7.2 × 10-10 M, respectively. Furthermore, L1Mab-352 and L1Mab-354 were applicable for the detection of dPD-L1 in immunohistochemical analysis in paraffin-embedded dPD-L1-overexpressed cells. These results indicated that L1Mab-352 and L1Mab-354 are useful for detecting dPD-L1 in immunohistochemical analysis.

Podocalyxin (PODXL) overexpression is associated with poor clinical outcomes in various tumors. PODXL is involved in tumor malignant progression through the promotion of invasiveness and metastasis. Therefore, PODXL is considered a promising target of monoclonal antibody (mAb)-based therapy. However, PODXL also plays an essential role in normal cells, such as vascular and lymphatic endothelial cells. Therefore, cancer specificity or selectivity is required to reduce adverse effects on normal cells. Here, we developed an anti-PODXL cancer-specific mAb (CasMab), PcMab-6 (IgG1, kappa), by immunizing mice with a soluble PODXL ectodomain derived from a glioblastoma LN229 cell. PcMab-6 reacted with the PODXL-positive LN229 cells but not with PODXL-knockout LN229 cells in flow cytometry. Importantly, PcMab-6 recognized pancreatic ductal adenocarcinoma (PDAC) cell lines (MIA PaCa-2, Capan-2, and PK-45H) but did not react with normal lymphatic endothelial cells (LECs). In contrast, one of the non-CasMabs, PcMab-47, showed high reactivity to both the PDAC cell lines and LECs. Next, we engineered PcMab-6 into a mouse IgG2a-type (PcMab-6-mG2a) and a humanized IgG1-type (humPcMab-6) mAb and further produced the core fucose-deficient types (PcMab-6-mG2a-f and humPcMab-6-f, respectively) to potentiate the antibody-dependent cellular cytotoxicity (ADCC). Both PcMab-6-mG2a-f and humPcMab-6-f exerted ADCC and complement-dependent cellular cytotoxicity in the presence of effector cells and complements, respectively. In the PDAC xenograft model, both PcMab-6-mG2a-f and humPcMab-6-f exhibited potent antitumor effects. These results indicated that humPcMab-6-f could apply to antibody-based therapy against PODXL-expressing pancreatic cancers.

The CXC chemokine receptor 4 (CXCR4, CD184) is a member of the G protein-coupled receptor family that is expressed in most leukocytes. Overexpression of CXCR4 is associated with poor prognosis in not only hematopoietic malignancy but also solid tumors. Because CXCR4 is an attractive target for tumor therapy, reliable preclinical murine models using anti-CXCR4 monoclonal antibodies (mAbs) have been warranted. This study established a novel anti-mouse CXCR4 (mCXCR4) mAb using the Cell-Based Immunization and Screening method. Flow cytometric analysis showed that an anti-mCXCR4 mAb, Cx4Mab-1 (rat IgG2a, kappa), recognized mCXCR4-overexpressed Chinese hamster ovary-K1 (CHO/mCXCR4) cells and endogenously mCXCR4-expressing mouse myeloma P3X63Ag8U.1 (P3U1) cells. Furthermore, Cx4Mab-1 did not recognize mCXCR4-knockout P3U1 cells. The dissociation constants of Cx4Mab-1 for CHO/mCXCR4 and P3U1 were determined as 6.4 × 10-9 M and 2.3 × 10-9 M, respectively, indicating that Cx4Mab-1 possesses a high affinity to both endogenous and exogenous mCXCR4-expressing cells. These results indicate that Cx4Mab-1 could be a useful tool for preclinical mouse models.

By converting extracellular adenosine triphosphate to adenosine, CD39 is involved in adenosine metabolism. The extracellular adenosine plays a critical role in the immune suppression of the tumor microenvironment. Therefore, the inhibition of CD39 activity by monoclonal antibodies (mAbs) is thought to be one of the important strategies for tumor therapy. In this study, we developed novel mAbs for mouse CD39 (mCD39) using the Cell-Based Immunization and Screening (CBIS) method. One of the established anti-mCD39 mAbs, C39Mab-2 (rat IgG2a, lambda), reacted with mCD39-overexpressed Chinese hamster ovary-K1 (CHO/mCD39) and an endogenously mCD39-expressed cell line (SN36) by flow cytometry. The kinetic analysis using flow cytometry indicated that the dissociation constant (KD) values of C39Mab-2 for CHO/mCD39 and SN36 were 5.5 × 10-9 M and 4.9 × 10-9 M, respectively. These results indicated that C39Mab-2 is useful for the detection of mCD39 in flow cytometry.

In small animal models of severe acute respiratory syndrome coronaviruses (SARS-CoV and SARS-CoV-2) infection, ferrets (Mustela putorius furo) have been used to investigate the pathogenesis. Podoplanin (PDPN) is an essential marker in lung type I alveolar epithelial cells, kidney podocytes, and lymphatic endothelial cells. Monoclonal antibodies (mAbs) against ferret PDPN (ferPDPN) are useful for the pathological analyses of those tissues. We previously established an anti-ferPDPN mAb, PMab-292 using the Cell-Based Immunization and Screening (CBIS) method. In this study, we determined the critical epitope of PMab-292 using flow cytometry. The ferPDPN deletion mutants analysis revealed that the Val34 is located at the N-terminus of the PMab-292 epitope. Furthermore, the PA tag-substituted analysis (PA scanning) showed that Asp39 is located at the C-terminus of PMab-292 epitope. The epitope sequence (VRPEDD) also exists between Val26 and Asp31 of ferPDPN, indicating that PMab-292 recognizes the tandem repeat of the VRPEDD sequence of ferPDPN.

Abstract Breast cancer patients with high levels of human epidermal growth factor receptor 2 (HER2) expression have worse clinical outcomes. Anti‐HER2 monoclonal antibody (mAb) is the most important therapeutic modality for HER2‐positive breast cancer. We previously immunized mice with the ectodomain of HER2 to create the anti‐HER2 mAb, H₂Mab‐77 (mouse IgG₁, kappa). This was then altered to produce H₂Mab‐77‐mG_2a‐f, an afucosylated mouse IgG_2a. In the present work, we examined the reactivity of H₂Mab‐77‐mG_2a‐f and antitumor effects against breast cancers in vitro and in vivo. BT‐474, an endogenously HER2‐expressing breast cancer cell line, was identified by H₂Mab‐77‐mG_2a‐f with a strong binding affinity (a dissociation constant [K_D]: 5.0 × 10⁻⁹ M). H₂Mab‐77‐mG_2a‐f could stain HER2 of breast cancer tissues in immunohistochemistry and detect HER2 protein in Western blot analysis. Furthermore, H₂Mab‐77‐mG_2a‐f demonstrated strong antibody‐dependent cellular cytotoxicity (ADCC) and complement‐dependent cytotoxicity (CDC) for BT‐474 cells. MDA‐MB‐468, a HER2‐negative breast cancer cell line, was unaffected by H₂Mab‐77‐mG_2a‐f. Additionally, in the BT‐474‐bearing tumor xenograft model, H₂Mab‐77‐mG_2a‐f substantially suppressed tumor development when compared with the control mouse IgG_2a mAb. In contrast, the HER2‐negative MDA‐MB‐468‐bearing tumor xenograft model showed no response to H₂Mab‐77‐mG_2a‐f. These findings point to the possibility of H₂Mab‐77‐mG_2a‐f as a treatment regimen by showing that it has antitumor effects on HER2‐positive breast tumors.

A cancer-specific anti-PDPN mAb, LpMab-23 (mouse IgG1, kappa), was established in our previous study. We herein produced a humanized IgG1 version (humLpMab-23) and defucosylated form (humLpMab-23-f) of an anti-PDPN mAb to increase ADCC activity. humLpMab-23 recognized PDPN-overexpressed Chinese hamster ovary (CHO)-K1 (CHO/PDPN), PDPN-positive PC-10 (human lung squamous cell carcinoma), and LN319 (human glioblastoma) cells via flow cytometry. We then demonstrated that humLpMab-23-f induced ADCC and complement-dependent cytotoxicity against CHO/PDPN, PC-10, and LN319 cells in vitro and exerted high antitumor activity in mouse xenograft models, indicating that humLpMab-23-f could be useful as an antibody therapy against PDPN-positive lung squamous cell carcinomas and glioblastomas.

The clinically approved human epidermal growth factor receptor 2 (HER2)-targeting monoclonal antibodies (mAbs), trastuzumab, and pertuzumab, target domains IV and II, respectively. Trastuzumab is now the standard treatment for HER2-overexpressed breast and gastric cancers, and trastuzumab in combination with pertuzumab showed clinical benefit. However, there still exist patients who do not respond to the therapy. Furthermore, HER2 mutants that cannot be recognized by pertuzumab were found in tumors. Therefore, novel anti-HER2 mAbs and modalities have been desired. In our previous study, we developed a novel anti-HER2 domain I mAb, H2Mab-139 (mouse IgG1, kappa). We herein produced a defucosylated mouse IgG2a type of mAb against HER2 (H2Mab-139-mG2a-f) to enhance antibody-dependent cellular cytotoxicity (ADCC)-mediated antitumor activity. H2Mab-139-mG2a-f exhibits a high binding affinity in flow cytometry with the dissociation constant (KD) determined to be 3.9 × 10-9 M and 7.7 × 10-9 M against HER2-overexpressed Chinese hamster ovary (CHO)-K1 (CHO/HER2) and HER2-positive BT-474 cells, respectively. Moreover, we showed that H2Mab-139-mG2a-f exerted ADCC and complement-dependent cytotoxicity against CHO/HER2 and BT-474 in vitro and exhibited potent antitumor activities in mouse xenograft models. These results indicated that H2Mab-139-mG2a-f exerts antitumor effects against HER2-positive human breast cancers and is useful as an antibody treatment for HER2-positive human cancers.

Carcinoma cells possess high proliferative and invasive potentials and exhibit a resilience against stresses, metabolic disorder, and therapeutic efforts. These properties are mainly acquired by genetic alterations including driver gene mutations. However, the detailed molecular mechanisms have not been fully elucidated. Here, we provide a novel mechanism connecting oncogenic signaling and the tumorigenic properties by a transforming growth factor-β1-stimulated clone 22 (TSC-22) family protein, THG-1 (also called as TSC22D4). THG-1 is localized at the basal layer of normal squamous epithelium and overexpressed in squamous cell carcinomas (SCCs). THG-1 knockdown suppressed SCC cell proliferation, invasiveness, and xenograft tumor formation. In contrast, THG-1 overexpression promoted the EGF-induced proliferation and stratified epithelium formation. Furthermore, THG-1 is phosphorylated by the receptor tyrosine kinase (RTK)-RAS-ERK pathway, which promoted the oncogene-mediated tumorigenesis. Moreover, THG-1 involves in the alternative splicing of CD44 variants, a regulator of invasiveness, stemness, and oxidative stress resistance under the RTK pathway. These findings highlight the pivotal roles of THG-1 as a novel effector of SCC tumorigenesis, and the detection of THG-1 phosphorylation by our established specific antibody could contribute to cancer diagnosis and therapy.

Overexpression of human epidermal growth factor receptor 2 (HER2) in breast cancer is an important target of monoclonal antibody (mAb) therapy such as trastuzumab. Due to the development of trastuzumab–deruxtecan, an antibody-drug conjugate, the targetable HER2-positive breast cancer patients have been expanded. To evaluate the developing modalities using anti-HER2 mAbs, reliable preclinical mouse models are required. Therefore, sensitive mAbs against mouse HER2 (mHER2) should be established. This study developed anti-mHER2 mAbs using the Cell-Based Immunization and Screening (CBIS) method. The established anti-mHER2 mAbs, H2Mab-300 (rat IgG2b, kappa) and H2Mab-304 (rat IgG1, kappa), reacted with mHER2-overexpressed Chinese hamster ovary-K1 (CHO/mHER2) and endogenously mHER2-expressed cell line, NMuMG (a mouse mammary gland epithelial cell) via flow cytometry. Furthermore, these mAbs never recognized mHER2-knockout NMuMG cells. The kinetic analysis using flow cytometry indicated that the dissociation constant (KD) values of H2Mab-300 and H2Mab-304 for CHO/mHER2 were 1.2 × 10−9 M and 1.7 × 10−9 M, respectively. The KD values of H2Mab-300 and H2Mab-304 for NMuMG were 4.9 × 10−10 M and 9.0 × 10−10 M, respectively. These results indicated that H2Mab-300 and H2Mab-304 could apply to the detection of mHER2 using flow cytometry and may be useful to obtain the proof of concept in preclinical studies.

Gastric cancer (GC) is the third leading cause of cancer-related deaths worldwide. GC with peritoneal metastasis exhibits a poor prognosis due to the lack of effective therapy. A comprehensive analysis of malignant ascites identified the genomic alterations and significant amplifications of cancer driver genes, including CD44. CD44 and its splicing variants are overexpressed in tumors, and play crucial roles in the acquisition of invasiveness, stemness, and resistance to treatments. Therefore, the development of CD44-targeted monoclonal antibodies (mAbs) is important for GC diagnosis and therapy. In this study, we immunized mice with CD44v3–10-overexpressed PANC-1 cells and established several dozens of clones that produce anti-CD44v3–10 mAbs. One of the clones (C44Mab-94; IgG1, kappa) recognized the variant-8-encoded region and peptide, indicating that C44Mab-94 is a specific mAb for CD44v8. Furthermore, C44Mab-94 could recognize CHO/CD44v3–10 cells, oral squamous cell carcinoma cell line (HSC-3), or GC cell lines (MKN45 and NUGC-4) in flow cytometric analyses. C44Mab-94 could detect the exogenous CD44v3–10 and endogenous CD44v8 in western blotting and stained the formalin-fixed paraffin-embedded gastric cancer cells. These results indicate that C44Mab-94 is useful for detecting CD44v8 in a variety of experimental methods and is expected to become usefully applied to GC diagnosis and therapy.

Epidermal Growth Factor Receptor (EGFR) overexpression or its mutation mediates the sustaining proliferative signaling, which is an important hallmark of cancer. Human EGFR-targeting monoclonal antibody (mAb) therapy such as cetuximab has been approved for clinical use in patients with colorectal cancers and head and neck squamous cell carcinomas. A reliable preclinical mouse model is essential to further develop the mAb therapy against EGFR. Therefore, sensitive mAbs against mouse EGFR (mEGFR) should be established. In this study, we developed a specific and sensitive mAb for mEGFR using the Cell-Based Immunization and Screening (CBIS) method. The established anti-mEGFR mAb, EMab-300 (rat IgG1, kappa), reacted with mEGFR-overexpressed Chinese hamster ovary-K1 (CHO/mEGFR) and endogenously mEGFR-expressed cell lines, including NMuMG (a mouse mammary gland epithelial cell) and Lewis lung carcinoma cells, using flow cytometry. The kinetic analysis using flow cytometry indicated that the KD of EMab-300 for CHO/mEGFR and NMuMG was 4.3 × 10−8 M and 1.9 × 10−8 M, respectively. These results indicated that EMab-300 applies to the detection of mEGFR using flow cytometry and may be useful to obtain the proof of concept in preclinical studies.

Head and neck squamous cell carcinoma (HNSCC) is the most common type of head and neck cancer, and has been revealed as the second-highest expression of CD44 in cancers. CD44 has been investigated as a cancer stem cell marker of HNSCC and plays a critical role in tumor malignant progression. Especially, splicing variant isoforms of CD44 (CD44v) are overexpressed in cancers and considered a promising target for cancer diagnosis and therapy. We developed monoclonal antibodies (mAbs) against CD44 by immunizing mice with CD44v3–10-overexpressed PANC-1 cells. Among the established clones, C44Mab-18 (IgM, kappa) reacted with CHO/CD44v3–10, but not with CHO/CD44s and parental CHO-K1 using flow cytometry. The epitope mapping using peptides that cover variant exon-encoded regions revealed that C44Mab-18 recognized the border sequence between variant 10 and the constant exon 16-encoded sequence. These results suggest that C44Mab-18 recognizes variant 10-containing CD44v, but not CD44s. Furthermore, C44Mab-18 could recognize the human oral squamous cell carcinoma (OSCC) cell line, HSC-3, in flow cytometry. The apparent dissociation constant (KD) of C44Mab-18 for CHO/CD44v3–10 and HSC-3 was 1.6 × 10−7 M and 1.7 × 10−7 M, respectively. Furthermore, C44Mab-18 detected CD44v3–10 but not CHO/CD44s in Western blotting, and endogenous CD44v10 in immunohistochemistry using OSCC tissues. These results indicate that C44Mab-18 is useful for detecting CD44v10 in flow cytometry and immunohistochemistry.

Cluster of differentiation 44 (CD44) promotes tumor progression through the recruitment of growth factors and the acquisition of stemness, invasiveness, and drug resistance. CD44 has multiple isoforms including CD44 standard (CD44s) and CD44 variants (CD44v), which have common and unique functions in tumor development. Therefore, elucidating the function of each CD44 isoform in a tumor is essential for the establishment of CD44-targeting tumor therapy. We have established various anti-CD44s and anti-CD44v monoclonal antibodies (mAbs) through the immunization of CD44v3-10-overexpressed cells. In this study, we established C44Mab-6 (IgG1, kappa), which recognized the CD44 variant 3-encoded region (CD44v3), as determined via an enzyme-linked immunosorbent assay. C44Mab-6 reacted with CD44v3-10-overexpressed Chinese hamster ovary (CHO)-K1 cells (CHO/CD44v3-10) or some cancer cell lines (COLO205 and HSC-3) via flow cytometry. The apparent KD of C44Mab-6 for CHO/CD44v3-10, COLO205, and HSC-3 was 1.5 × 10-9 M, 6.3 × 10-9 M, and 1.9 × 10-9 M, respectively. C44Mab-6 could detect the CD44v3-10 in Western blotting and stained the formalin-fixed paraffin-embedded tumor sections in immunohistochemistry. These results indicate that C44Mab-6 is useful for detecting CD44v3 in various experiments and is expected for the application of tumor diagnosis and therapy.

Pancreatic cancer exhibits a poor prognosis due to the lack of early diagnostic biomarkers and the resistance to conventional chemotherapy. CD44 has been known as a cancer stem cell marker and plays tumor promotion and drug resistance roles in various cancers. In particular, the splicing variants are overexpressed in many carcinomas and play essential roles in the cancer stemness, invasiveness or metastasis, and resistance to treatments. Therefore, the understanding of each CD44 variant's (CD44v) function and distribution in carcinomas is essential for the establishment of CD44-targeting tumor therapy. In this study, we immunized mice with CD44v3-10-overexpressed Chinese hamster ovary (CHO)-K1 cells and established various anti-CD44 monoclonal antibodies (mAbs). One of the established clones (C44Mab-3; IgG1, kappa) recognized peptides of the variant-5-encoded region, indicating that C44Mab-3 is a specific mAb for CD44v5. Moreover, C44Mab-3 reacted with CHO/CD44v3-10 cells or pancreatic cancer cell lines (PK-1 and PK-8) by flow cytometry. The apparent KD of C44Mab-3 for CHO/CD44v3-10 and PK-1 was 1.3 × 10-9 M and 2.6 × 10-9 M, respectively. C44Mab-3 could detect the exogenous CD44v3-10 and endogenous CD44v5 in Western blotting and stained the formalin-fixed paraffin-embedded pancreatic cancer cells but not normal pancreatic epithelial cells in immunohistochemistry. These results indicate that C44Mab-3 is useful for detecting CD44v5 in various applications and is expected to be useful for the application of pancreatic cancer diagnosis and therapy.

CC chemokine receptor 6 (CCR6) is one of the members of the G-protein-coupled receptor (GPCR) family that is upregulated in many immune-related cells, such as B lymphocytes, effector and memory T cells, regulatory T cells, and immature dendritic cells. The coordination between CCR6 and its ligand CC motif chemokine ligand 20 (CCL20) is deeply involved in the pathogenesis of various diseases, such as cancer, psoriasis, and autoimmune diseases. Thus, CCR6 is an attractive target for therapy and is being investigated as a diagnostic marker for various diseases. In a previous study, we developed an anti-mouse CCR6 (mCCR6) monoclonal antibody (mAb), C6Mab-13 (rat IgG1, kappa), that was applicable for flow cytometry by immunizing a rat with the N-terminal peptide of mCCR6. In this study, we investigated the binding epitope of C6Mab-13 using an enzyme-linked immunosorbent assay (ELISA) and the surface plasmon resonance (SPR) method, which were conducted with respect to the synthesized point-mutated-peptides within the 1-20 amino acid region of mCCR6. In the ELISA results, C6Mab-13 lost its ability to react to the alanine-substituted peptide of mCCR6 at Asp11, thereby identifying Asp11 as the epitope of C6Mab-13. In our SPR analysis, the dissociation constants (KD) could not be calculated for the G9A and D11A mutants due to the lack of binding. The SPR analysis demonstrated that the C6Mab-13 epitope comprises Gly9 and Asp11. Taken together, the key binding epitope of C6Mab-13 was determined to be located around Asp11 on mCCR6. Based on the epitope information, C6Mab-13 could be useful for further functional analysis of mCCR6 in future studies.

Cluster of differentiation 44 (CD44) is a type I transmembrane glycoprotein and has been shown to be a cell surface marker of cancer stem-like cells in various cancers. In particular, the splicing variants of CD44 (CD44v) are overexpressed in cancers and play critical roles in cancer stemness, invasiveness, and resistance to chemotherapy and radiotherapy. Therefore, the understanding of the function of each CD44v is indispensable for CD44-targeting therapy. CD44v9 contains the variant 9-encoded region, and its expression predicts poor prognosis in patients with various cancers. CD44v9 plays critical roles in the malignant progression of tumors. Therefore, CD44v9 is a promising target for cancer diagnosis and therapy. Here, we developed sensitive and specific monoclonal antibodies (mAbs) against CD44 by immunizing mice with CD44v3-10-overexpressed Chinese hamster ovary-K1 (CHO/CD44v3-10) cells. We first determined their critical epitopes using enzyme-linked immunosorbent assay and characterized their applications as flow cytometry, western blotting, and immunohistochemistry. One of the established clones, C44Mab-1 (IgG1, kappa), reacted with a peptide of the variant 9-encoded region, indicating that C44Mab-1 recognizes CD44v9. C44Mab-1 could recognize CHO/CD44v3-10 cells or colorectal cancer cell lines (COLO201 and COLO205) in flow cytometric analysis. The apparent dissociation constant (KD) of C44Mab-1 for CHO/CD44v3-10, COLO201, and COLO205 was 2.5 × 10-8 M, 3.3 × 10-8 M, and 6.5 × 10-8 M, respectively. Furthermore, C44Mab-1 was able to detect the CD44v3-10 in western blotting and the endogenous CD44v9 in immunohistochemistry using colorectal cancer tissues. These results indicated that C44Mab-1 is useful for detecting CD44v9 not only in flow cytometry or western blotting but also in immunohistochemistry against colorectal cancers.

One of G protein-coupled receptors, CC chemokine receptor 3 (CCR3), is expressed in eosinophils, basophils, a subset of Th2 lymphocytes, mast cells, and airway epithelial cells. CCR3 levels in the serum of colorectal cancer patients are significantly higher than in control groups. Moreover, CCR3 is essential for recruiting eosinophils into the lung. Therefore, CCR3 is considered both a therapeutic target for colorectal cancer and allergic diseases. Previously, we established anti-mouse CCR3 (mCCR3) monoclonal antibodies (mAbs), C3Mab-6 (rat IgG1, kappa) and C3Mab-7 (rat IgG1, kappa), by immunizing a rat with an N-terminal peptide of mCCR3. These mAbs can be used in flow cytometry and enzyme-linked immunosorbent assays. In this study, we performed the epitope mapping of C3Mab-6 and C3Mab-7 using alanine scanning. The reactivity between these mAbs and point mutants of mCCR3 were analyzed using flow cytometry. The results indicated that Phe3, Asn4, Thr5, Asp6, Glu7, Lys9, Thr10, and Glu13 of mCCR3 are essential for C3Mab-6 binding, whereas Phe15 and Glu16 are essential for C3Mab-7 binding.

Cluster of differentiation 44 (CD44) has been investigated as a cancer stem cell (CSC) marker as it plays critical roles in tumor malignant progression. The splicing variants are overexpressed in many carcinomas, especially squamous cell carcinomas, and play critical roles in the promotion of tumor metastasis, the acquisition of CSC properties, and resistance to treatments. Therefore, each CD44 variant (CD44v) function and distribution in carcinomas should be clarified for the establishment of novel tumor diagnosis and therapy. In this study, we immunized mouse with a CD44 variant (CD44v3-10) ectodomain and established various anti-CD44 monoclonal antibodies (mAbs). One of the established clones (C44Mab-34; IgG1, kappa) recognized a peptide that covers both variant 7- and variant 8-encoded regions, indicating that C44Mab-34 is a specific mAb for CD44v7/8. Moreover, C44Mab-34 reacted with CD44v3-10-overexpressed Chinese hamster ovary-K1 (CHO) cells or the oral squamous cell carcinoma (OSCC) cell line (HSC-3) by flow cytometry. The apparent KD of C44Mab-34 for CHO/CD44v3-10 and HSC-3 was 1.4 × 10-9 and 3.2 × 10-9 M, respectively. C44Mab-34 could detect CD44v3-10 in Western blotting and stained the formalin-fixed paraffin-embedded OSCC in immunohistochemistry. These results indicate that C44Mab-34 is useful for detecting CD44v7/8 in various applications and is expected to be useful in the application of OSCC diagnosis and therapy.

We recently developed a novel anti-human C-C chemokine receptor 9 (hCCR9) monoclonal antibody (mAb), C9Mab-11, which is applicable to flow cytometry, western blotting, and enzyme-linked immunosorbent assay (ELISA). This study aims to identify the binding epitope of C9Mab-11 by using 1 × and 2 × alanine (or glycine) substituted-hCCR9 peptides (1 × and 2 × Ala-scan) by ELISA. According to the 1 × Ala-scan analysis, the response of C9Mab-11 was diminished against M13A of the hCCR9 peptide, but was not eliminated. In the 2 × Ala-scan analysis, the reactions were abolished in the substitution of P11A-N12A, N12A-M13A, and M13A-A14G of hCCR9 N-terminal peptides. The results indicate that the binding epitope of C9Mab-11 includes Pro11, Asn12, Met13, and Ala14 of hCCR9, with the region around Met13 being particularly important. The successful identification of the C9Mab-11 epitope might be useful for the future pathophysiological analysis of hCCR9.

CD44 has been known as a marker of tumor-initiating cells, and plays pro-tumorigenic functions in many cancers. The splicing variants play critical roles in the malignant progression of cancers by promoting stemness, cancer cell invasion or metastasis, and resistance to chemo- and radiotherapy. To understand each CD44 variant (CD44v) function is essential to know the property of cancers and the establishment of the therapy. However, the function of the variant 4-encoded region has not been elucidated. Therefore, specific monoclonal antibodies (mAbs) against variant 4 are indispensable for basic research, tumor diagnosis, and therapy. In this study, we established anti-CD44 variant 4 (CD44v4) mAbs by immunizing mice with a peptide containing the variant 4-encoded region. We next performed flow cytometry, western blotting, and immunohistochemistry to characterize them. One of the established clones (C44Mab-108; IgG1, kappa) reacted with CD44v3-10-overexpressed Chinese hamster ovary-K1 cells (CHO/CD44v3-10). The KD of C44Mab-108 for CHO/CD44 v3-10 was 3.4 × 10-7 M. In western blot analysis, C44Mab-108 detected CD44v3-10 in the lysate of CHO/CD44v3-10 cells. Furthermore, C44Mab-108 stained formalin-fixed paraffin-embedded (FFPE) oral squamous carcinoma tissues in immunohistochemistry. These results indicated that C44Mab-108 is useful to detect CD44v4 in immunohistochemistry using FFPE tissues.

CD44 is a cell surface glycoprotein, and its isoforms are produced by the alternative splicing with the standard and variant exons. The CD44 variant exon-containing isoforms (CD44v) are overexpressed in carcinomas. CD44v6 is one of the CD44v, and its overexpression predicts poor prognosis in colorectal cancer (CRC) patients. CD44v6 plays critical roles in CRC adhesion, proliferation, stemness, invasiveness, and chemoresistance. Therefore, CD44v6 is a promising target for cancer diagnosis and therapy for CRC. In this study, we established anti-CD44 monoclonal antibodies (mAbs) by immunizing mice with CD44v3-10-overexpressed Chinese hamster ovary (CHO)-K1 cells. We then characterized them using enzyme-linked immunosorbent assay, flow cytometry, western blotting, and immunohistochemistry. One of the established clones (C44Mab-9; IgG1, kappa) reacted with a peptide of the variant 6-encoded region, indicating that C44Mab-9 recognizes CD44v6. Furthermore, C44Mab-9 reacted with CHO/CD44v3-10 cells or CRC cell lines (COLO201 and COLO205) by flow cytometry. The apparent dissociation constant (KD) of C44Mab-9 for CHO/CD44v3-10, COLO201, and COLO205 was 8.1 × 10-9 M, 1.7 × 10-8 M, and 2.3 × 10-8 M, respectively. C44Mab-9 detected the CD44v3-10 in western blotting, and partially stained the formalin-fixed paraffin-embedded CRC tissues in immunohistochemistry. Collectively, C44Mab-9 is useful for detecting CD44v6 in various applications.

The epithelial cell adhesion molecule (EpCAM) is a type I transmembrane glycoprotein, and plays critical roles in cell adhesion, proliferation, and tumorigenesis. EpCAM has been considered as a promising target for tumor diagnosis and therapy. Anti-EpCAM monoclonal antibodies (mAbs) have been developed for EpCAM-overexpressed tumors, and several clinical trials have demonstrated promising outcomes. We previously established an anti-EpCAM mAb, EpMab-37 (mouse IgG1, kappa), using the Cell-Based Immunization and Screening method. EpMab-37 was revealed to recognize the conformational epitope of EpCAM. In this study, we determined the critical epitope of EpMab-37 by flow cytometry using the 1 × alanine scanning (1 × Ala-scan) and the 2 × alanine scanning (2 × Ala-scan) method. We first performed flow cytometry by 1 × Ala-scan using one alanine (or glycine)-substituted EpCAM mutants, which were expressed on Chinese hamster ovary-K1 cells, and found that the EpMab-37 did not recognize the R163A mutant of EpCAM. We next performed flow cytometry by 2 × Ala-scan using two alanine (or glycine) residues-substituted EpCAM mutants, and confirmed that EpMab-37 did not recognize R163A-including mutants of EpCAM. The results indicated that the critical binding epitope of EpMab-37 includes Arg163 of EpCAM.

Epithelial cell adhesion molecule (EpCAM) is a type I transmembrane glycoprotein, which is highly expressed on tumor cells. As EpCAM plays a crucial role in cell adhesion, survival, proliferation, stemness, and tumorigenesis, it has been considered as a promising target for tumor diagnosis and therapy. Anti‑EpCAM monoclonal antibodies (mAbs) have been developed and have previously demonstrated promising outcomes in several clinical trials. An anti‑EpCAM mAb, EpMab‑37 (mouse IgG1, kappa) was previously developed by the authors, using the cell‑based immunization and screening method. In the present study, a defucosylated version of anti‑EpCAM mAb (EpMab‑37‑mG2a‑f) was generated to evaluate the antitumor activity against EpCAM‑positive cells. EpMab‑37‑mG2a‑f recognized EpCAM‑overexpressing CHO‑K1 (CHO/EpCAM) cells with a moderate binding‑affinity [dissociation constant (KD)=2.2x10‑8 M] using flow cytometry. EpMab‑37‑mG2a‑f exhibited potent antibody‑dependent cellular cytotoxicity (ADCC) and complement‑dependent cytotoxicity (CDC) for CHO/EpCAM cells by murine splenocytes and complements, respectively. Furthermore, the administration of EpMab‑37‑mG2a‑f significantly suppressed CHO/EpCAM xenograft tumor development compared with the control mouse IgG. EpMab‑37‑mG2a‑f also exhibited a moderate binding‑affinity (KD=1.5x10‑8 M) and high ADCC and CDC activities for a colorectal cancer cell line (Caco‑2 cells). The administration of EpMab‑37‑mG2a‑f to Caco‑2 tumor‑bearing mice significantly suppressed tumor development compared with the control. By contrast, EpMab‑37‑mG2a‑f never suppressed the xenograft tumor growth of Caco‑2 cells in which EpCAM was knocked out. On the whole, these results indicate that EpMab‑37‑mG2a‑f may exert antitumor activities against EpCAM‑positive cancers and may thus be a promising therapeutic regimen for colorectal cancer.

C-C chemokine receptor 9 (CCR9) is a receptor for C-C-chemokine ligand 25 (CCL25). CCR9 is crucial in the chemotaxis of immune cells and inflammatory responses. Moreover, CCR9 is highly expressed in tumors, including several solid tumors and T-cell acute lymphoblastic leukemia. Several preclinical studies have shown that anti-CCR9 monoclonal antibodies (mAbs) exert antitumor activity. Therefore, CCR9 is an attractive target for tumor therapy. In this study, we conducted the epitope mapping of an anti-mouse CCR9 (mCCR9) mAb, C9Mab-24 (rat IgG2a, kappa), using the 1× alanine (1× Ala)- and 2× alanine (2× Ala)-substitution methods via enzyme-linked immunosorbent assay. We first performed the 1× Ala-substitution method using one alanine-substituted peptides of the mCCR9 N-terminus (amino acids 1-19). C9Mab-24 did not recognize two peptides (F14A and F17A), indicating that Phe14 and Phe17 are critical for C9Mab-24-binding to mCCR9. Furthermore, we conducted the 2× Ala-substitution method using two consecutive alanine-substituted peptides of the mCCR9 N-terminus, and showed that C9Mab-24 did not react with four peptides (M13A-F14A, F14A-D15A, D16A-F17A, and F17A-S18A), indicating that 13-MFDDFS-18 is involved in C9Mab-24-binding to mCCR9. Overall, combining, the 1× Ala- or 2× Ala-scanning methods could be useful for understanding for target-antibody interaction.

The CC chemokine receptor 3 (CCR3) is a receptor for CC chemokines, including CCL5/RANTES, CCL7/MCP-3, and CCL11/eotaxin. CCR3 is expressed on the surface of eosinophils, basophils, a subset of Th2 lymphocytes, mast cells, and airway epithelial cells. CCR3 and its ligands are involved in airway hyperresponsiveness in allergic asthma, ocular allergies, and cancers. Therefore, CCR3 is an attractive target for those therapies. Previously, anti-mouse CCR3 (mCCR3) monoclonal antibodies (mAbs), C3Mab-3 (rat IgG2a, kappa), and C3Mab-4 (rat IgG2a, kappa) were developed using the Cell-Based Immunization and Screening (CBIS) method. In this study, the binding epitope of these mAbs was investigated using flow cytometry. A CCR3 extracellular domain-substituted mutant analysis showed that C3Mab-3, C3Mab-4, and a commercially available mAb (J073E5) recognized the N-terminal region (amino acids 1–38) of mCCR3. Next, alanine scanning was conducted in the N-terminal region. The results revealed that the Ala2, Phe3, Asn4, and Thr5 of mCCR3 are involved in C3Mab-3 binding, whereas Ala2, Phe3, and Thr5 are essential to C3Mab-4 binding, and Ala2 and Phe3 are crucial to J073E5 binding. These results reveal the involvement of the N-terminus of mCCR3 in the recognition of C3Mab-3, C3Mab-4, and J073E5.

The epithelial cell adhesion molecule (EpCAM) is a stem cell and carcinoma antigen, which mediates cellular adhesion and proliferative signaling by the proteolytic cleavage. In contrast to low expression in normal epithelium, EpCAM is frequently overexpressed in various carcinomas, which correlates with poor prognosis. Therefore, EpCAM has been considered as a promising target for tumor diagnosis and therapy. Using the Cell-Based Immunization and Screening (CBIS) method, we previously established an anti-EpCAM monoclonal antibody (EpMab-37; mouse IgG1, kappa). In this study, we investigated the antibody-dependent cellular cytotoxicity (ADCC), complement-dependent cytotoxicity (CDC), and an antitumor activity by a defucosylated mouse IgG2a-type of EpMab-37 (EpMab-37-mG2a-f) against a breast cancer cell line (BT-474) and a pancreatic cancer cell line (Capan-2), both of which express EpCAM. EpMab-37-mG2a-f recognized BT-474 and Capan-2 cells with a moderate binding-affinity [apparent dissociation constant (KD): 2.9 × 10−8 M and 1.8 × 10−8 M, respectively] by flow cytometry. EpMab-37-mG2a-f exhibited ADCC and CDC for both cells by murine splenocytes and complements, respectively. Furthermore, administration of EpMab-37-mG2a-f significantly suppressed the xenograft tumor development compared with the control mouse IgG. These results indicated that EpMab-37-mG2a-f exerts antitumor activities and could provide valuable therapeutic regimen for breast and pancreatic cancers.

Human epidermal growth factor receptor 2 (HER2) has been studied in many human cancer types, and its overexpression and/or gene mutation contribute to the poor prognosis. Therefore, HER2 is an important therapeutic target in various cancer types, including breast and gastric cancers. We previously developed an anti-HER2 monoclonal antibody (mAb), H2Mab-77 (mouse IgG1, kappa), which detects HER2 and dog HER2 (dHER2) with high sensitivity and specificity. In this study, we produced a defucosylated mouse-dog chimeric anti-HER2 mAb (H77Bf), and investigated the reactivity against canine osteosarcoma D-17 cells by flow cytometry. Furthermore, we showed that H77Bf exerted antibody-dependent cellular cytotoxicity and complement-dependent cytotoxicity against D-17 cells in vitro and exhibited the potent antitumor activity in vivo. These results suggest that H77Bf exerts antitumor effects against dHER2-expressing canine tumors and could be valuable as part of an antibody treatment regimen for them.

The overexpression of epidermal growth factor receptors (EGFRs) has been reported in various human tumors, including breast, gastric, lung, colorectal, and pancreatic cancers. Humanized anti-EGFR and anti-human epidermal growth factor receptor 2 (HER2) monoclonal antibodies (mAbs) have been shown to improve patients’ survival. Canine tumors resemble human tumors in the initiation and progression. We previously established a defucosylated mouse-dog chimeric anti-EGFR mAb (E134Bf) and a mouse-dog chimeric anti-HER2 mAb (H77Bf), which exerted antitumor activities in canine tumor xenograft models. Here, we produced E134Bf antibody fused to H77Bf single chain Fv at the light chains (E134Bf-H77scFv). The bispecific E134Bf-H77scFv recognized dog EGFR (dEGFR) and dog HER2 (dHER2)-overexpressed Chinese hamster ovary-K1 cells by flow cytometry. E134Bf-H77scFv also reacted with dEGFR/dHER2-positive canine osteosarcoma D-17 cells, and possesses a high binding-affinity (KD: 1.3 × 10−9 M). Furthermore, E134Bf-H77scFv exerted antibody-dependent cellular cytotoxicity and complement-dependent cytotoxicity against D-17 cells in the presence of canine mononuclear cells and complement, respectively. Moreover, administration of E134Bf-H77scFv suppressed the development of D-17 xenograft tumor in mice early compared with the control dog IgG, E134Bf and H77Bf alone. These results indicate that E134Bf-H77scFv exerts antitumor activities against dEGFR/dHER2-positive canine tumors, and could be a valuable treatment regimen for canine tumors.

The C-C chemokine receptor 9 (CCR9) belongs to the G-protein-coupled receptor superfamily, and is highly expressed on the T cells and intestinal cells. CCR9 regulates various immune responses by binding to the C-C chemokine ligand, CCL25, and is involved in inflammatory diseases and tumors. Therefore, the development of sensitive monoclonal antibodies (mAbs) for CCR9 is necessary for treatment and diagnosis. In this study, we established a specific anti-human CCR9 (hCCR9) mAb; C9Mab-11 (mouse IgG2a, kappa), using the synthetic peptide immunization method. C9Mab-11 reacted with hCCR9-overexpressed Chinese hamster ovary-K1 (CHO/hCCR9) and hCCR9-endogenously expressed MOLT-4 (human T-lymphoblastic leukemia) cells in flow cytometry. The dissociation constant (KD) of C9Mab-11 for CHO/hCCR9 and MOLT-4 cells were determined to be 1.2 × 10-9 M and 4.9 × 10-10 M, respectively, indicating that C9Mab-11 possesses a high affinity for both exogenously and endogenously hCCR9-expressing cells. Furthermore, C9Mab-11 clearly detected hCCR9 protein in CHO/hCCR9 cells using western blot analysis. In summary, C9Mab-11 can be a useful tool for analyzing hCCR9-related biological responses.

The CC chemokine receptor 6 (CCR6) is a G protein-coupled receptor family member that is highly expressed in B lymphocytes, certain subsets of effector and memory T cells, and immature dendritic cells. CCR6 has only one chemokine ligand, CCL20. The CCL20-CCR6 axis has been recognized as a therapeutic target for autoimmune diseases and tumor. This study developed specific monoclonal antibodies (mAbs) against mouse CCR6 (mCCR6) using the peptide immunization method. The established anti-mCCR6 mAb, C6Mab-13 (rat IgG1, kappa), reacted with mCCR6-overexpressed Chinese hamster ovary-K1 (CHO/mCCR6), and mCCR6-endogenously expressed P388 (mouse lymphoid neoplasma) and J774-1 (mouse macrophage-like) cells in flow cytometry. The dissociation constant (KD) of C6Mab-13 for CHO/mCCR6 cells was determined to be 2.8 × 10-9 M, indicating that C6Mab-13 binds to mCCR6 with high affinity. In summary, C6Mab-13 is useful for detecting mCCR6-expressing cells through flow cytometry.

CC chemokine receptor type-2 (CCR2) is a member of the G protein-coupled receptors, and is mainly expressed on cell surface of immune cells. CCR2 binds to its ligand, C-C motif chemokine 2 (also named as monocyte chemoattractant protein-1), which involves in the tumor progression by modulating the tumor microenvironment. Therefore, the monoclonal antibody (mAb) targeting CCR2 could be one of the strategies for cancer treatment. In this study, we investigated the critical epitope of C2Mab-6, an anti-mouse CCR2 (mCCR2) mAb developed by N-terminal peptides immunization. We first performed enzyme-linked immunosorbent assay (ELISA) using N-terminal peptides of mCCR2 and demonstrated that C2Mab-6 recognizes 1-19 amino acids of mCCR2. We further performed ELISA using 20 alanine-substituted peptides of mCCR2. C2Mab-6 lost the reaction to the alanine-substituted peptides of D3A, N4A, M6A, P8A, Q9A, and F10A. These results indicate that the binding epitope of C2Mab-6 includes Asp3, Asn4, Met6, Pro8, Gln9, and Phe10 of mCCR2.

CC chemokine receptor type-2 (CCR2) belongs to the G protein-coupled receptors superfamily, and is localized on cell surface of tumor cells and some immune cells, including monocytes and macrophages. CCR2 is a receptor for monocyte chemoattractant protein-1/C-C motif chemokine 2, and is involved in the progression of various diseases such as cancers. Therefore, the development of CCR2-targeted monoclonal antibody (mAb) is desired. Its characterization, including epitope of mAb, is very important for antibody applications. In this study, we investigated the critical epitope of K036C2, which is a commercially available anti-human CCR2 (hCCR2) mAb. We conducted enzyme-linked immunosorbent assay (ELISA) using three N-terminal peptides of hCCR2 and demonstrated that K036C2 recognizes 11-29 and 21-39 amino acids of hCCR2. We further performed ELISA using 20 peptides, which include alanine substitution of hCCR2. K036C2 lost the reaction to the alanine-substituted peptides of D25A, Y26A, D27A, G29A, and A30G. These results indicate that the critical binding epitope of K036C2 includes Asp25, Tyr26, Asp27, Gly29, and Ala30 of hCCR2.

The CXC chemokine receptor 6 (CXCR6) is a member of the G protein-coupled receptor family that is highly expressed in helper T type 1 cells, cytotoxic T lymphocytes (CTLs), and natural killer cells. CXCR6 plays critical roles in local expansion of effector-like CTLs in tumor microenvironment to potentiate the antitumor response. Therefore, the development of anti-CXCR6 monoclonal antibodies (mAbs) is essential to evaluate the immune microenvironment of tumors. Using N-terminal peptide immunization, we previously developed an anti-mouse CXCR6 (mCXCR6) mAb, Cx6Mab-1 (rat IgG1, kappa) , which is useful for flow cytometry and western blotting. In this study, we determined the critical epitope of Cx6Mab-1 by enzyme-linked immunosorbent assay (ELISA) using the 1 × alanine scanning (1 × Ala-scan) method or the 2 × alanine scanning (2 × Ala-scan) method. Although we first performed ELISA by 1 × Ala-scan using one alanine-substituted peptides of mCXCR6 N-terminal domain (amino acids 1-20), we could not identify the Cx6Mab-1 epitope. We next performed ELISA by 2 × Ala-scan using two alanine (or glycine) residues-substituted peptides of mCXCR6 N-terminal domain, and found that Cx6Mab-1 did not recognize S8A-A9G, A9G-L10A, L10A-Y11A, and G13A-H14A of the mCXCR6 N-terminal peptide. The results indicate that the binding epitope of Cx6Mab-1 includes Ser8, Ala9, Leu10, Tyr11, Gly13, and His14 of mCXCR6. Therefore, we could demonstrate that the 2 × Ala scan method is useful for determining the critical epitope of mAbs.

Immune checkpoint molecules have received attention as targets of cancer immunotherapy. Killer cell lectin-like receptor subfamily G member 1 (KLRG1) is one of the immune checkpoint molecules expressed in CD4+ T, CD8+ T, and natural killer (NK) cells. KLRG1 exhibits antiviral and antitumor immunity, and its expression in T and NK cells is upregulated by viral infectious diseases and some tumors. Thus, monoclonal antibodies (mAbs) for KLRG1 would be useful tools for the diagnosis and immunotherapy against viral infectious diseases and cancers. We have developed anti-human KLRG1 (hKLRG1) mAb (clone KLMab-1, mouse IgG1, kappa) by the Cell-Based Immunization and Screening method. We have also demonstrated that KLMab-1 recognizes both exogenous and endogenous hKLRG1 in flow cytometry. In this study, we first showed that KLMab-1 and its recombinant mAb (recKLMab-1) bound to exogenous hKLRG1 overexpressed in Chinese hamster ovary (CHO)-K1 cells, but not in parental CHO-K1 cells, in immunocytochemistry. We next showed that both mAbs detected endogenous hKLRG1 expressed in human NK cells. These results demonstrate that KLMab-1 and recKLMab-1 are available for immunocytochemistry.

Human epidermal growth factor receptor 2 (HER2) overexpression has been reported in various types of cancer, including breast, gastric, lung, colorectal and pancreatic cancer. A humanized anti‑HER2 monoclonal antibody (mAb), trastuzumab, has been shown to improve survival of patients in HER2‑positive breast and gastric cancer. An anti‑HER2 mAb, H2Mab‑77 (mouse IgG1, kappa) was previously developed. In the present study, a defucosylated version of mouse‑dog chimeric anti‑HER2 mAb (H77Bf) was generated. H77Bf possesses a high binding‑affinity [a dissociation constant (KD): 7.5x10‑10 M, as determined by flow cytometric analysis] for dog HER2‑overexpressed CHO‑K1 (CHO/dHER2) cells. H77Bf highly exerted antibody‑dependent cellular cytotoxicity (ADCC) and complement‑dependent cytotoxicity (CDC) for CHO/dHER2 cells by canine mononuclear cells and complement, respectively. Moreover, administration of H77Bf significantly suppressed the development of CHO/dHER2 xenograft tumor in mice compared with the control dog IgG. H77Bf also possesses a high binding‑affinity (KD: 7.2x10‑10 M) for a canine mammary gland tumor cell line (SNP), and showed high ADCC and CDC activities for SNP cells. Intraperitoneal administration of H77Bf in mouse xenograft models of SNP significantly suppressed the development of SNP xenograft tumors compared with the control dog IgG. These results indicated that H77Bf exerts antitumor activities against dHER2‑positive canine cancers, and could be valuable treatment regimen for canine cancers.

Podoplanin (PDPN) is a marker of lung type I alveolar cells, kidney podocytes, and lymphatic endothelial cells. The overexpression of PDPN contributes to the malignant progression of tumors. Therefore, the development of anti-PDPN monoclonal antibodies (mAbs) to animals is essential to evaluate the pathogenesis and cellular functions. Using peptide immunization, we previously developed an anti-elephant PDPN (elePDPN) mAb, PMab-295, which is useful for flow cytometry, Western blotting, and immunohistochemistry. In this study, we determined the critical epitope of PMab-295 by enzyme-linked immunosorbent assay (ELISA). We performed ELISA with the alanine-substituted peptides of elePDPN extracellular domain (amino acids 38-51), and found that PMab-295 did not recognize the alanine-substituted peptides of M41A, P44A, and E47A. Furthermore, these peptides could not inhibit the recognition of PMab-295 to elePDPN-expressing cells by flow cytometry and immunohistochemistry. The results indicate that the binding epitope of PMab-295 includes Met41, Pro44, and Glu47 of elePDPN.

Podoplanin (PDPN) is an essential marker of lung type I alveolar cells, kidney podocytes, and lymphatic endothelial cells. Monoclonal antibodies (mAbs) that can specifically recognize PDPN in immunohistochemistry are important to analyze the development of tissues and the pathogenesis of diseases, including cancers. We have developed anti-PDPN mAbs against many animal species; however, mAbs that can recognize elephant-derived membrane proteins and distinguish the specific cell types in immunohistochemistry are limited. In this study, a novel anti-elephant PDPN (elePDPN) mAb, PMab-295 (IgG1, kappa), was established using the peptide immunization method. PMab-295 recognized both elePDPN-overexpressed Chinese hamster ovary (CHO)-K1 cells and endogenous elePDPN-expressed LACF-NaNaI cells by flow cytometry and western blotting. Kinetic analyses using flow cytometry showed that the KD of PMab-295 for CHO/elePDPN was 1.5 × 10-8 M. Furthermore, PMab-295 detected elePDPN-expressing cells using immunohistochemistry. These results showed the usefulness of PMab-295 to investigate the molecular function of elePDPN and the pathogenesis of diseases.

The CC chemokine receptor type-2 (CCR2) is one of the members of the G protein-coupled receptor superfamily, which are expressed on the cell surface of immune and tumor cells. CCR2 binds to the C-C motif chemokine ligand 2 (CCL2)/monocyte chemoattractant protein-1 (MCP-1), which is produced by various cells, including tumor and immune-related cells. Therefore, the development of sensitive monoclonal antibodies (mAbs) for CCR2 has been desired for treatment and diagnosis. In this study, we established a specific antihuman CCR2 (hCCR2) mAb, C2Mab-9 (mouse IgG1, kappa), using the synthetic peptide immunization method. Flow cytometric and immunocytochemical results showed that C2Mab-9 reacted with hCCR2-expressing U937 (human histiocytic lymphoma) and natural killer cells. Furthermore, C2Mab-9 showed the moderate binding affinity for both cells. Conclusively, C2Mab-9 can be a useful tool for analyzing hCCR2-related biological responses.

C-C chemokine receptor 4 (CCR4) is one of G protein-coupled receptors, and interacts with chemokines, CCL17 and CCL22. CCR4 is expressed on T cells such as helper T type 2 cells, regulatory T cells, and interleukin 17-producing T helper cells. CCR4 is associated with T cells trafficking into the tumor microenvironment, and is associated with tumor progression or metastasis. Therefore, CCR4 may be a potential therapeutic option for T cell malignancies. C4Mab-1 is a novel anti-mouse CCR4 (mCCR4) monoclonal antibody produced by mCCR4 N-terminal peptide immunization. C4Mab-1 is useful for flow cytometric analysis. In this study, we conducted the epitope mapping of C4Mab-1 using enzyme-linked immunosorbent assay (ELISA) and peptide blocking assay. The result of ELISA indicated that Thr7, Asp8, and Gln11 of mCCR4 are the critical amino acids for the C4Mab-1 binding. Furthermore, peptide blocking assay by flow cytometry showed that Thr7, Asp8, and Gln11 of mCCR4 are essential for C4Mab-1 binding to mCCR4-overexpressed Chinese hamster ovary-K1 (CHO/mCCR4) cells, and Val6, Thr9, and Thr10 are involved in the C4Mab-1 binding to CHO/mCCR4 cells. These results indicate that the critical binding epitope of C4Mab-1 includes Thr7, Asp8, and Gln11 of mCCR4.

The epithelial cell adhesion molecule (EpCAM) is a cell surface glycoprotein, which is widely expressed on normal and cancer cells. EpCAM is involved in cell adhesion, proliferation, survival, stemness, and tumorigenesis. Therefore, EpCAM is thought to be a promising target for cancer diagnosis and therapy. In this study, we established anti-EpCAM monoclonal antibodies (mAbs) using the Cell-Based Immunization and Screening (CBIS) method. We characterized them using flow cytometry, Western blotting, and immunohistochemistry. One of the established recombinant anti-EpCAM mAbs, recEpMab-37 (mouse IgG1, kappa), reacted with EpCAM-overexpressed Chinese hamster ovary-K1 cells (CHO/EpCAM) or a colorectal carcinoma cell line (Caco-2). In contrast, recEpMab-37 did not react with EpCAM-knocked out Caco-2 cells. The KD of recEpMab-37 for CHO/EpCAM and Caco-2 was 2.0 × 10-8 M and 3.2 × 10-8 M, respectively. We observed that EpCAM amino acids between 144 to 164 are involved in recEpMab-37 binding. In Western blot analysis, recEpMab-37 detected the EpCAM of CHO/EpCAM and Caco-2 cells. Furthermore, recEpMab-37 could stain formalin-fixed paraffin-embedded colorectal carcinoma tissues by immunohistochemistry. Taken together, recEpMab-37, established by the CBIS method, is useful for detecting EpCAM in various applications.

CC chemokine receptor type-2 (CCR2) belongs to the G protein-coupled receptors superfamily, localized on cell surface of some immune-related cells, including monocytes and macrophages. CCR2 and its ligand CCL2 are involved in the progression of various diseases such as cancers. Therefore, CCR2-targeted monoclonal antibodies (mAbs) are needed for treatment and diagnosis. Previously, we successfully developed an anti-human CCR2 (hCCR2) mAb, C2Mab-9 (mouse IgG1, kappa), which is applicable for flow cytometry and immunocytochemistry. In this study, we investigated the critical epitope of C2Mab-9. We conducted enzyme-linked immunosorbent assay (ELISA) using several N-terminal peptides of hCCR2, and demonstrated that C2Mab-9 recognizes 11-29 and 21-39 amino acids of hCCR2. We further performed ELISA using 20 peptides, which include alanine substitution of hCCR2. C2Mab-9 lost the reaction to the alanine-substituted peptides of F23A, F24A, D25A, Y26A, and D27A. Among them, F23A, F24A, D25A, and Y26A did not block the C2Mab-9 reaction with U937 cells in flow cytometry. These results indicate that the critical binding epitope of C2Mab-9 includes Phe23, Phe24, Asp25, and Tyr26.

Chinese hamster (Cricetulus griseus) and golden hamster (Mesocricetus auratus) are important animal models of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infections, which affect several organs, including respiratory tract, lung, and kidney. Podoplanin (PDPN) is a marker of lung type I alveolar cells, kidney podocytes, and lymphatic endothelial cells. The development of anti-PDPN monoclonal antibodies (mAbs) for these animals is essential to evaluate the pathogenesis by SARS-CoV-2 infections. Using the Cell-Based Immunization and Screening method, we previously developed an anti-Chinese hamster PDPN (ChamPDPN) mAb, PMab-281 (mouse IgG3, kappa), and further changed its subclass into IgG2a (281-mG2a-f), both of which can recognize not only ChamPDPN but also golden hamster PDPN (GhamPDPN) by flow cytometry and immunohistochemistry. In this study, we examined the critical epitope of 281-mG2a-f, using enzyme-linked immunosorbent assay (ELISA) with synthesized peptides. First, we performed ELISA with peptides derived from ChamPDPN and GhamPDPN extracellular domain, and found that 281-mG2a-f reacted with the peptides, which commonly possess the KIPFEELxT sequence. Next, we analyzed the reaction with the alanine-substituted mutants, and revealed that 281-mG2a-f did not recognize the alanine-substituted peptides of I75A, F77A, and E79A of ChamPDPN. Furthermore, these peptides could not inhibit the recognition of 281-mG2a-f to ChamPDPN-expressing cells by flow cytometry. The results indicate that the binding epitope of 281-mG2a-f includes Ile75, Phe77, and Glu79 of ChamPDPN, which are shared with GhamPDPN.

The epidermal growth factor receptor (EGFR) contributes to tumor malignancy through gene amplification and/or protein overexpression. In our previous study, we developed an anti-human EGFR (hEGFR) monoclonal antibody, clone EMab-134 (mouse IgG1, kappa), which specifically detects both hEGFR and dog EGFR (dEGFR). The defucosylated mouse IgG2a version of EMab-134 (134-mG2a-f) exhibits antibody-dependent cellular cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC) in dEGFR-overexpressed Chinese hamster ovary-K1 (CHO/dEGFR) cells and antitumor activities in mouse xenografts of CHO/dEGFR cells. In this study, the reactivity of 134-mG2a-f against a canine mammary gland tumor cell line (SNP) was examined by flow cytometry and immunocytochemistry. Furthermore, 134-mG2a-f highly exerted ADCC and CDC for SNP. The administration of 134-mG2a-f significantly suppressed the SNP xenograft growth. These results suggest that 134-mG2a-f exerts antitumor effects against dEGFR-expressing canine mammary gland tumors, and could be valuable as part of an antibody treatment regimen for them.

The CXC chemokine receptor 6 (CXCR6) is a member of the G protein-coupled receptor family that is highly expressed in helper T type 1 cells, natural killer cells, cytotoxic T lymphocytes, and various type of cells in tumor microenvironment (TME). CXCR6 has been proposed as a therapeutic target against tumors through regulation of the tumor TME. In this study, we developed specific and sensitive monoclonal antibodies (mAbs) for mouse CXCR6 (mCXCR6), which are useful for flow cytometry and Western blotting by N-terminal peptide immunization into rat. The established anti-mCXCR6 mAb, Cx6Mab-1 (rat IgG1, kappa), reacted with not only mCXCR6-overexpressed Chinese hamster ovary-K1 (CHO/mCXCR6) but also mCXCR6-endogenously expressed cell lines, such as P388 (mouse lymphoid neoplasm) and J774-1 (mouse macrophage-like) through flow cytometry. Kinetic analyses using flow cytometry indicated that the dissociation constants (KD) of Cx6Mab-1 for CHO/mCXCR6, P388, and J774-1 cells were 1.7 × 10-9 M, 3.4 × 10-7 M, and 3.8 × 10-7 M, respectively. Furthermore, Cx6Mab-1 could detect endogenous mCXCR6 in P388 and J774-1 cells by Western blotting. These results indicated that Cx6Mab-1 is useful for detecting mCXCR6 by flow cytometry and Western blotting, and provides a possibility for targeting CXCR6-expressing cells in vivo experiments.

CD44 is a cell surface glycoprotein, which is expressed on normal cells, and overexpressed on cancer cells. CD44 is involved in cell adhesion, migration, proliferation, survival, stemness, and chemo-resistance. Therefore, CD44 is thought to be a promising target for cancer diagnosis and therapy. In this study, we established anti-CD44 monoclonal antibodies (mAbs) by immunizing mice with a CD44 variant (CD44v3-10) ectodomain and screening using enzyme-linked immunosorbent assay. We then characterized them using flow cytometry, Western blotting, and immunohistochemistry. One of the established clones (C44Mab-46; IgG1, kappa) reacted with CD44 standard isoform (CD44s)-overexpressed Chinese hamster ovary-K1 cells (CHO/CD44s) or esophageal squamous cell carcinoma (ESCC) cell lines (KYSE70 and KYSE770). The apparent KD of C44Mab-46 for CHO/CD44s, KYSE70, and KYSE770 was 1.1 × 10-8 M, 4.9 × 10-8 M, and 4.1 × 10-8 M, respectively. C44Mab-46 detected CD44s of CHO/CD44s and KYSE70, and CD44 variants of KYSE770 in Western blot analysis. Furthermore, C44Mab-46 strongly stained the formalin-fixed paraffin-embedded ESCC tissues in immunohistochemistry. Collectively, C44Mab-46 is very useful for detecting CD44 in various applications.

CD10 is a cell surface metalloendopeptidase that cleaves and degrades many secreted physiologically active peptides by its enzymatic activity. Although CD10 expression has been found in various types of cells, its expression is increased in several cancers, including renal cancer. In this study, the antitumor activity of a novel anti-human CD10 monoclonal antibody (mAb) was investigated. A defucosylated mouse IgG2a version of C10Mab-31 (31-mG2a-f) was created from an anti-CD10 mAb, C10Mab-31 (IgG1, kappa). Both C10Mab-31 and 31-mG2a-f specifically reacted with endogenous CD10 in renal cancer cells, VMRC-RCW, with the dissociation constant (KD) values of 6.3 × 10-9 M and 1.1 × 10-9 M, respectively, indicating high binding affinity. To further examine the anti-CD10 mAb-mediated effector functions, the antibody-dependent cellular cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC) were examined. The 31-mG2a-f significantly exhibited ADCC and CDC against VMRC-RCW cells in vitro. Furthermore, 31-mG2a-f exhibited antitumor activities in mouse xenografts of VMRC-RCW cells. These results suggest that 31-mG2a-f exerts antitumor activities against CD10-expressing renal cancers and could be a valuable therapeutic candidate for treating them.

It has been widely accepted that monoclonal antibody (mAb) is an effective tool for cancer immunotherapy. The C-C motif chemokine receptor 8 (CCR8) is highly expressed in regulatory T cells and many cancers and is associated with the progression of the cancers. However, its role in cancer progression remains unclear. Thus, the development of mAbs for CCR8 leads to cancer immunotherapy and elucidation of unknown mechanisms of CCR8-dependent cancer progression. In this study, we have developed an anti-mouse CCR8 (mCCR8) mAb (clone C8Mab-1, rat IgG2a, kappa) using the Cell-Based Immunization and Screening (CBIS) method. We showed that C8Mab-1 and its recombinant antibody (recC8Mab-1) bind to mCCR8-overexpressed Chinese hamster ovary (CHO)-K1 cells (CHO/mCCR8), but not to the parental CHO-K1 cells, in flow cytometry and immunofluorescence. Moreover, C8Mab-1 and recC8Mab-1 specifically reacted to P388 (a mouse lymphocyte-like cells) and J774-1 (a mouse macrophage-like cells), which express endogenous mCCR8, in both applications. These results suggest that C8Mab-1, developed using the CBIS method, is useful for flow cytometry and immunocytochemistry against exogenous and endogenous mCCR8.

Golden (Syrian) hamster (Mesocricetus auratus) is a small animal model of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infections. Pathological analyses of the tissues are required to understand the pathogenesis of SARS-CoV-2 and the evaluation of therapeutic modalities, including neutralizing monoclonal antibodies (mAbs). However, mAbs that recognize the golden hamster-derived antigens and distinguish specific cell types, such as the pneumocytes, are limited. Podoplanin (PDPN) is an essential marker of lung type I alveolar epithelial cells, kidney podocytes, and lymphatic endothelial cells. In this study, an anti-Chinese hamster (Cricetulus griseus) PDPN mAb PMab-281 (IgG3, kappa) was established using the Cell-Based Immunization and Screening (CBIS) method. A defucosylated mouse IgG2a version of PMab-281 (281-mG2a-f) was also developed. The 281-mG2a-f strongly recognized both the Chinese hamster and the golden hamster PDPN using flow cytometry and could detect lung type I alveolar epithelial cells, lymphatic endothelial cells, and Bowman's capsules in the kidney from the golden hamster using immunohistochemistry. These results suggest the usefulness of 281-mG2a-f for analyzing the golden hamster-derived tissues and cells for SARS-CoV-2 research.

The C-C motif chemokine receptor 8 (CCR8) is highly expressed in regulatory T cells. CCR8 is also expressed in many cancers and is associated with those progression. The development of monoclonal antibodies (mAbs) for CCR8 leads to cancer immunotherapy and elucidation of unknown mechanisms of CCR8-dependent cancer progression. In this study, we have developed an anti-mouse CCR8 (mCCR8) mAb (clone C8Mab-3, rat IgG1, kappa) using the Cell-Based Immunization and Screening (CBIS) method. We revealed that C8Mab-3 and its recombinant antibody (recC8Mab-3) bind to mCCR8-overexpressed Chinese hamster ovary (CHO)-K1 cells (CHO/mCCR8), but not to the parental CHO-K1 cells, in flow cytometry. In addition, C8Mab-3 and recC8Mab-3 reacted to P388 (a mouse lymphocyte-like cell) and J774-1 (a mouse macrophage-like cell), which express endogenous mCCR8. C8Mab-3 also detected exogenous and endogenous mCCR8 using immunocytochemistry. These results suggest that C8Mab-3, developed using the CBIS method, is useful for immunocytochemistry against exogenous and endogenous mCCR8.

The epidermal growth factor receptor (EGFR) is involved in tumor malignancy through gene amplification and/or protein overexpression. An anti-human EGFR (hEGFR) monoclonal antibody (clone EMab-134), which explicitly detects hEGFR and dog EGFR (dEGFR), was previously developed. The defucosylated mouse IgG2a version of EMab-134 (134-mG2a-f) exhibits antibody-dependent cellular cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC) in dEGFR-overexpressed CHO-K1 (CHO/dEGFR) cells and antitumor activities in mouse xenografts of CHO/dEGFR cells. In this study, it was shown that 134-mG2a-f reacts with a canine fibroblastic tumor cell line (A-72) using flow cytometry and immunocytochemistry. Furthermore, 134-mG2a-f exerted ADCC and CDC on A-72 cell line. The administration of 134-mG2a-f significantly inhibited the A-72 xenograft growth. These results suggest that 134-mG2a-f exerts antitumor effects on dEGFR-expressing canine fibroblastic tumors.

The CC chemokine receptor type-2 (CCR2) belongs to the G-protein-coupled receptor superfamily, expressed on the cell surface of immune cells and tumors. CCR2 binds to the CC motif chemokine 2/monocyte chemoattractant protein-1, a CC chemokine, which is produced by various cells, including immune-related cells and tumors. Therefore, the development of sensitive monoclonal antibodies (mAbs) for CCR2 has been desired for treatment and diagnosis. This study established a novel, specific, and sensitive anti-mouse CCR2 (mCCR2) mAb; C2Mab-6 (rat IgG1, kappa), using the mCCR2 synthetic peptide immunization method. C2Mab-6 reacted with mCCR2-overexpressed Chinese hamster ovary-K1 cells and L1210 (murine leukemia) cells, which express endogenous mCCR2 in flow cytometry. Furthermore, C2Mab-6 showed a high binding affinity for both cells. Hence, C2Mab-6 can be a useful tool for analyzing mCCR2-related biological responses, using flow cytometry.

The CC chemokine receptor type-4 (CCR4) belongs to the G-protein-coupled receptor superfamily, expressed on the cell surface of T cells and its malignancy. Two CCR4 ligands (CCL17 and CCL22) bind to CCR4 that mediate physiological and pathological functions of T cell immune responses. Anti-CCR4 monoclonal antibody (mAb) mogamulizumab is approved for adult T cell leukemia/lymphoma and cutaneous T cell lymphomas. In addition, mogamulizumab can deplete regulatory T cells, implying the application to solid tumors as an immunomodulator. Therefore, the development of sensitive mAbs for CCR4 has been desired for basic research, diagnosis, and therapy. In this study, a specific, and sensitive anti-mouse CCR4 (mCCR4) mAb, C4Mab-1 (rat IgG1, kappa), was established using N-terminal peptide immunization. C4Mab-1 reacted with mCCR4-overexpressed Chinese hamster ovary (CHO)-K1 cells, P388 (mouse lymphoid neoplasm), and J774-1 (mouse macrophage-like) cells in flow cytometry. Kinetic analyses using flow cytometry showed that KDs of C4Mab-1 for CHO/mCCR4, P388, and J774-1 cells were 4.2 × 10-9 M, 5.4 × 10-7 M, and 1.1 × 10-6 M, respectively. C4Mab-1 could be a valuable tool for elucidating mCCR4-related biological responses.

C-C motif chemokine receptor 9 (CCR9) is a G protein-coupled receptor, which is highly expressed in T-lymphocytes and different cancer cells. CCR9 aggravates immune diseases and cancer progression and is considered a biomarker and a therapeutic target of diseases. The development of specific monoclonal antibody (mAbs) for human CCR9 (hCCR9) is required to diagnose and treat immune diseases and cancers. Previously, we established the cell-based immunization and screening (CBIS) method, which does not need purified target proteins. Anti-hCCR9 mAb (clone C9Mab-1; mouse IgG1, kappa) was also developed using the CBIS method. C9Mab-1 is usable for flow cytometry against exogenously and endogenously expressing hCCR9. This study showed that C9Mab-1 and its recombinant antibody (recC9Mab-1) specifically detected exogenous hCCR9 stably overexpressed in Chinese hamster ovary (CHO)-K1 cells and endogenous hCCR9 expressed in a human T-lymphoblastic leukemia cell line MOLT-4 cells through immunocytochemistry. This study provides a new application of C9Mab-1 and recC9Mab-1 in immunocytochemistry.

The epidermal growth factor receptor (EGFR) contributes to tumor malignancy through gene amplification and/or protein overexpression. In our previous study, we developed an anti-human EGFR (hEGFR) monoclonal antibody (mAb), clone EMab-134 (mouse IgG1, kappa), which specifically detects both hEGFR and dog EGFR (dEGFR). The defucosylated mouse IgG2a version of EMab-134 exhibits antibody-dependent cellular cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC) in dEGFR-overexpressed CHO-K1 (CHO/dEGFR) cells and antitumor activities in mouse xenografts of CHO/dEGFR cells. In this study, we produced a defucosylated mouse-dog chimeric anti-EGFR mAb (E134Bf), and the reactivity of E134Bf against a canine mammary gland tumor cell line (SNP) was examined by flow cytometry. Furthermore, E134Bf highly exerted ADCC and CDC for SNP cells. The administration of E134Bf with canine mononuclear cells significantly suppressed the SNP xenograft growth. These results suggest that E134Bf exerts antitumor effects against dEGFR-expressing canine mammary gland tumors and could be valuable as part of an antibody treatment regimen for them.

CC chemokine receptor 3 (CCR3) belongs to the G protein-coupled receptor family and is highly expressed in eosinophils and basophils. CCR3 is essential for recruiting eosinophils into the lung. Moreover, CCR3 was found in the serum of colorectal cancer patients higher than in the control group. Therefore, CCR3 will be a useful target for asthma and colorectal cancer diagnosis. This study developed a specific and sensitive monoclonal antibody (mAb) for mouse CCR3 (mCCR3), which is useful for flow cytometry using the Cell-Based Immunization and Screening method. The established anti-mCCR3 mAb, C3Mab-3 (rat IgG2a, kappa), reacted with mCCR3-overexpressed Chinese hamster ovary-K1 (CHO/mCCR3) cells through flow cytometry. C3Mab-3 also reacted with P388 (mouse lymphoid neoplasma) and J774-1 (mouse macrophage-like) cells, which express mCCR3 endogenously. Kinetic analyses using flow cytometry indicated that KDs of C3Mab-3 for CHO/mCCR3, P388, and J774-1 cells were 4.3 × 10-8 M, 2.6 × 10-7 M, and 2.4 × 10-7 M, respectively. C3Mab-3 could be a valuable tool for elucidating mCCR3-related biological response using flow cytometry.

CD10 is a glycosylated transmembrane protein and is known as a membrane endopeptidase. It is expressed on predifferentiated lymphocyte progenitor, epithelial, stromal, and tumor cells. Therefore, antibodies against CD10 are used for diagnosing follicular lymphoma and solid tumors, including renal carcinomas. In this study, we developed an anti-human CD10 monoclonal antibody, clone C10Mab-31 (IgG1, kappa), which detects CD10 by flow cytometry and shows high affinity for CD10-overexpressed CHO-K1 (CHO/CD10) cells. Furthermore, the defucosylated mouse IgG2a version of C10Mab-31 (31-mG2a-f) exhibits antibody-dependent cellular cytotoxicity, complement-dependent cytotoxicity, and antitumor activities in mouse xenografts of CHO/CD10 cells. These results indicate that 31-mG2a-f exerts antitumor effects against CD10-expressing tumors and could be valuable as part of an antibody treatment regimen for them.

C-C motif chemokine receptor 8 (CCR8) is a G protein-coupled receptor predominantly expressed in regulatory T (Treg) and T helper 2 cells. The evidence that CCR8 expression in Treg is increased in cancers, CCR8 increases migration activity of Treg, and CCR8 induces the anti-apoptotic activity in T cell leukemia and lymphoma suggests that CCR8 is associated with cancer development. Thus, developing a specific monoclonal antibody (mAb) for CCR8 is useful for diagnostic and therapeutic purposes and the anti-CCR8 mAb becomes a remarkable experimental tool for basic research. We previously developed an anti-mouse CCR8 (mCCR8) mAb called C8Mab-2 (rat IgG2b, kappa) that was applicable to flow cytometric analysis for both endogenous and exogenous mCCR8. This study showed that C8Mab-2 and recombinant C8Mab-2 (recC8Mab-2) were specifically bound to exogenously expressed mCCR8 in mCCR8-overexpressed Chinese hamster ovary-K1 cells. In addition, we found that C8Mab-2 and recC8Mab-2 recognized endogenous mCCR8 in P388 (a mouse lymphocyte-like cell line) and J774-1 cells (a mouse macrophage-like cell line). These data demonstrate that C8Mab-2 and recC8Mab-2 are useful for immunocytochemical analysis.

Ferrets (Mustela putorius furo) have been used as small animal models to investigate severe acute respiratory syndrome coronaviruses (SARS-CoV and SARS-CoV-2) infections. Pathological analyses of these tissue samples, including those of the lung, are, therefore, essential to understand the pathogenesis of SARS-CoVs and evaluate the action of therapeutic monoclonal antibodies (mAbs) against this disease. However, mAbs that recognize ferret-derived proteins and distinguish between specific cell types, such as lung epithelial cells, are limited. Podoplanin (PDPN) has been identified as an essential marker in lung type I alveolar epithelial cells, kidney podocytes, and lymphatic endothelial cells. In this study, an anti-ferret PDPN (ferPDPN) mAb PMab-292 (mouse IgG1, kappa) was established using the Cell-Based Immunization and Screening (CBIS) method. PMab-292 recognized ferPDPN-overexpressed Chinese hamster ovary-K1 (CHO/ferPDPN) cells by flow cytometry and Western blotting. The kinetic analysis using flow cytometry showed that the KD of PMab-292 for CHO/ferPDPN was 3.4 × 10-8 M. Furthermore, PMab-292 detected lung type I alveolar epithelial cells, lymphatic endothelial cells, and glomerular/Bowman's capsule in the kidney using immunohistochemistry. Hence, these results propose the usefulness of PMab-292 in analyzing ferret-derived tissues for SARS-CoV-2 research.

The CC chemokine receptor 3 (CCR3) is a member of the G protein-coupled receptor family that is highly expressed in eosinophils and basophils. CCR3 has been proposed as a therapeutic target for human immunodeficiency virus and allergy diagnosis. Therefore, in this study, we developed specific and sensitive monoclonal antibodies (mAbs) for mouse CCR3 (mCCR3), which are useful for flow cytometry by peptide immunization. The established anti-mCCR3 mAbs, C3Mab-6 (rat IgG1, kappa) and C3Mab-7 (rat IgG1, kappa), reacted with mCCR3-overexpressed Chinese hamster ovary-K1 (CHO/mCCR3), in addition to mCCR3-endogenously expressed cell lines, such as P388 (mouse lymphoid neoplasma) and J774-1 (mouse macrophage-like) through flow cytometry. Kinetic analyses using flow cytometry indicated that the dissociation constants (KDs) of C3Mab-6 for CHO/mCCR3, P388, and J774-1 cells were 8.7 × 10-9 M, 1.4 × 10-7 M, and 1.7 × 10-7 M, respectively, whereas the KDs of C3Mab-7 for these cell lines were 3.7 × 10-9 M, 5.1 × 10-7 M, and 3.1 × 10-7 M, respectively. Results also indicated that C3Mab-6 and C3Mab-7 are useful for detecting cells expressing CCR3 through flow cytometry, thereby making them potentially beneficial for treating CCR3-expressing cells.

Podoplanin (PDPN) is a cell-surface mucin-like glycoprotein that plays a critical role in tumor development and normal development of the lung, kidney, and lymphatic vascular systems. PDPN is overexpressed in several tumors and is involved in their malignancy. PDPN induces platelet aggregation through binding to platelet receptor C-type lectin-like receptor 2. Furthermore, PDPN modulates signal transductions that regulate cell proliferation, differentiation, migration, invasion, epithelial-to-mesenchymal transition, and stemness, all of which are crucial for the malignant progression of tumor. In the tumor microenvironment (TME), PDPN expression is upregulated in the tumor stroma, including cancer-associated fibroblasts (CAFs) and immune cells. CAFs play significant roles in the extracellular matrix remodeling and the development of immunosuppressive TME. Additionally, PDPN functions as a co-inhibitory molecule on T cells, indicating its involvement with immune evasion. In this review, we describe the mechanistic basis and diverse roles of PDPN in the malignant progression of tumors and discuss the possibility of the clinical application of PDPN-targeted cancer therapy, including cancer-specific monoclonal antibodies, and chimeric antigen receptor T technologies.

CD20, which is expressed on B lymphocytes, has been studied as a therapeutic target for B cell lymphomas and autoimmune disorders. Identifying the binding epitopes of monoclonal antibodies (mAbs) can contribute to our understanding of their functions. We have previously developed an anti-CD20 mAb (clone C20Mab-11) using a Cell-Based Immunization and Screening (CBIS) method. In this study, we aimed to determine the binding epitopes of anti-CD20 mAbs, such as C20Mab-11 and 2H7, using the His-tag insertion for epitope mapping (HisMAP). The results showed that 171-NPSE-174 and 168-EPANPSE-174 in the second loop of CD20 were essential for C20Mab-11 binding and 2H7 binding, respectively. Although we developed many mAbs that recognize conformational epitopes using the CBIS method, there are many difficulties in epitope mapping for these mAbs. HisMAP could be useful for determining the conformational epitopes of other mAbs against membrane proteins.

The C-C motif chemokine receptor 3 (CCR3) is a G protein-coupled receptor activated by eotaxin-1-3, MCP-2-4, and RANTES. CCR3 is associated with allergic diseases and cancer development and is highly expressed in eosinophils, basophils, and cancer cells. Besides, research on the physiological roles of CCR3 is ongoing. Thus, specific monoclonal antibodies (mAbs) for CCR3 would be useful for diagnostic and therapeutic purposes and for unraveling the function of CCR3. We previously developed an anti-mouse CCR3 (mCCR3) mAb (C3Mab-2; rat IgG2b, kappa) using the Cell-Based Immunization and Screening method and showed that C3Mab-2 could detect endogenous and exogenous mCCR3 in flow cytometry. In this study, we showed that C3Mab-2 and its recombinant antibody (recC3Mab-2f) specifically recognized endogenous mCCR3 in P388 (a mouse lymphocyte-like cell line) and J774-1 (a mouse macrophage-like cell line) cells and are usable in immunocytochemistry.

The epidermal growth factor receptor (EGFR) is a transmembrane glycoprotein. Although EGFR is physiologically essential in normal cells, it contributes to tumor malignancy through gene amplification and/or protein overexpression, which augment signaling cascades in tumor cells. We previously developed an anti-human EGFR (hEGFR) monoclonal antibody (mAb), EMab-134 (mouse IgG1, kappa), which detects hEGFR and dog EGFR (dEGFR) with high sensitivity and specificity. The mouse IgG2a version of EMab-134 (134-mG2a) has antitumor effects toward mouse xenografts of hEGFR-expressing oral squamous cell carcinomas. Furthermore, 134-mG2a-f, the defucosylated version of 134-mG2a, exhibits antibody-dependent cellular cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC) in dEGFR-overexpressed CHO-K1 (CHO/dEGFR) cells and antitumor activities in mouse xenografts of CHO/dEGFR cells. Herein, the reactivity of 134-mG2a-f against canine cancer cells with endogenous dEGFR was first examined by flow cytometry and immunocytochemistry. In vitro analysis demonstrated that 134-mG2a-f highly exerted ADCC and CDC for a canine osteosarcoma cell line, D-17, which expresses endogenous dEGFR. Moreover, in vivo administration of 134-mG2a-f significantly suppressed the development of D-17 compared with the results in response to control mouse IgG. These results suggest that 134-mG2a-f exerts antitumor effects against dEGFR-expressing canine cancers, and could be valuable as part of an antibody treatment regimen for them.

CD10 is a glycosylated transmembrane protein and is known as a membrane endopeptidase. CD10 is expressed on predifferentiated lymphocyte progenitor, epithelial, stromal, and tumor cells. Antibodies against CD10 are used for the diagnosis of follicular lymphoma. Anti-human CD10 monoclonal antibody (clone MME/1870) can be used for Western blotting and immunohistochemical analyses. This study examined the critical epitope of MME/1870 using enzyme-linked immunosorbent assay (ELISA) with synthesized peptides. First, we performed ELISA with deletion mutants, and MME/1870 reacted to the 501-520 amino acid sequence of CD10. Next, we analyzed the reaction to 20 point mutants, and MME/1870 did not recognize the alanine-substituted peptides of Y507A, I511A, I512A, and L515A. These results indicate that the binding epitope of MME/1870 includes Tyr507, Ile511, Ile512, and Leu515 of CD10.

Monoclonal antibodies (mAbs) that specifically target podoplanin (PDPN), a marker for type I alveolar cells, are required for immunohistochemical analyses. Anti-PDPN mAbs are available for many species, including human, mouse, rat, rabbit, dog, cat, bovine, pig, Tasmanian devil, alpaca, tiger, whale, goat, horse, bear, sheep, and California sea lion PDPNs. However, no anti-Steller sea lion PDPN (stePDPN) antibody has been developed. Immunohistochemical analysis showed that an anti-California sea lion PDPN mAb (PMab-269) reacted with type I alveolar cells from the Steller sea lion lung, renal glomeruli and Bowman's capsules from kidney, and lymphatic endothelial cells from the colon, indicating that PMab-269 is useful for detecting stePDPN.

CD20 is expressed in the B lymphocyte, and an effective target for the detection and treatment of B cell lymphomas. Therefore, CD20 has been studied as a therapeutic target of B cell lymphomas and autoimmune disorders. Specific anti-CD20 monoclonal antibodies (mAbs), such as rituximab, ofatumumab, veltuzumab, and ocaratuzumab, have been developed. Revealing the recognition mechanism of antigen by mAbs could contribute to understanding the function of mAbs and could be useful for the development of vaccine. Rituximab is a mouse-human chimeric anti-CD20 mAb, which was developed and approved for the treatment of the B cell malignancies. Hence, the binding epitope of rituximab for CD20 has been studied. Some reports show that 170-ANPS-173, especially Ala170 and Pro172 of CD20 are important for rituximab binding. However, only phage display results showed that 182-YCYSI-186 of CD20 is also important for rituximab binding to CD20. In this study, we tried to determine the binding epitope of rituximab for CD20 using histidine-tag insertion for epitope mapping (HisMAP) method. The results showed that two regions of CD20 (169-PANPSE-174 and 183-CYSIQ-187) are important for rituximab-binding for CD20.

Background: Human epidermal growth factor receptor 3 (HER3) has been found to be overexpressed in many cancers such as lung, breast, and colon cancers. High expression of HER3 is thought to be a negative prognostic factor in several solid tumors including colorectal cancer. Moreover, HER3 is associated with drug resistance to EGFR- and HER2-targeted therapy in some cancers. Therefore, HER3 is thought to be an important therapeutic target. Purpose: We aimed to develop a novel anti-HER3 monoclonal antibody (mAb), and investigated its antitumor activities against colorectal cancer. Methods: We employed the Cell-Based Immunization and Screening (CBIS) method using HER3-overexpressed CHO-K1 cells for producing anti-HER3 mAbs. Anti-HER3 mAbs were screened using flow cytometry (FCM). Then, we examined the antibody‑dependent cellular cytotoxicity (ADCC) and the complement‑dependent cytotoxicity (CDC) activities of anti-HER3 mAbs for Caco‑2 (a human colorectal adenocarcinoma cell line). Moreover, a mouse xenograft model of Caco-2 was used for examining the antitumor activity. Results: We developed an anti-HER3 mAbs, H₃Mab-17 (IgG_2a, kappa) using CBIS method. H₃Mab-17 reacted with HER3-expressing cells in FCM. In vitro analysis demonstrated that H₃Mab-17 showed the ADCC and CDC activities against Caco-2. H₃Mab-17 significantly reduced tumor development in Caco-2 xenograft compared with control mouse IgG. Conclusion: We have successfully established a novel anti-HER3 mAb (H₃Mab-17), which could be a useful antibody-based therapy for patients with HER3-expressing colorectal cancers.

CC motif chemokine receptor 8 (CCR8), a G protein-coupled receptor (GPCR), is highly expressed in regulatory T cells, T helper 2 cells, and cancer cells. It plays important role in allergic inflammation and cancer development. Therefore, specific monoclonal antibodies (mAbs) for CCR8 would be useful for diagnostic and therapeutic purposes of the diseases. However, the production of mAbs for GPCRs has remained very difficult. We have developed a novel method for the development of mAbs, named the Cell-Based Immunization and Screening (CBIS) method. In the present study, an SD rat was immunized with mouse CCR8-overexpressed CHO-K1 cells (CHO/mCCR8). The hybridomas expressing anti-mCCR8 mAbs were screened by using CHO/mCCR8 and CHO-K1 cells. We obtained 73 strongly anti-mCCR8 mAb-expressing hybridomas from 1,916 hybridomas, and we finally established C₈Mab-2 (IgG_2b, kappa). C₈Mab-2 selectively reacted to CHO/mCCR8 cells in a dose-dependent manner, but not to CHO-K1 cells, in flow cytometry. C₈Mab-2 also recognized endogenous mCCR8 in a mouse lymphocyte-like cell line (P388) and a mouse macrophage-like cell line (J774-1). Furthermore, C₈Mab-2 visualized mCCR8 in CHO/mCCR8, P388, and J774-1 cells in immunocytochemistry. In conclusion, we developed the anti-mCCR8 mAb, C₈Mab-2, which is available for detecting endogenous and exogenous mCCR8 in flow cytometry and immunocytochemistry. C₈Mab-2 would be usable for diagnosis and medical treatment for allergic inflammation and cancer in mouse models.

The epidermal growth factor receptor (EGFR) contributes to tumor malignancy via gene amplification and protein overexpression. Previously, we developed an anti-human EGFR (hEGFR) monoclonal antibody, EMab-134, which detects hEGFR and dog EGFR (dEGFR) with high sensitivity and specificity by flow cytometry, western blotting and immunohistochemistry. In this study, we produced a defucosylated mouse–dog chimeric anti-EGFR monoclonal antibody, E134Bf. Kinetic analysis of the interactions of E134Bf with the canine osteosarcoma cell line (D-17) and canine fibroblastic cell line (A-72) cells was conducted by flow cytometry. The K_D for the interaction of E134Bf with the D-17 and A-72 cells was 5.5 × 10⁻¹⁰ M and 6.0 × 10⁻¹⁰ M, respectively, indicating that E134Bf exhibits high affinity for D-17 and A-72 cells. Furthermore, E134Bf highly exerted antibody-dependent cellular cytotoxicity and complement-dependent cytotoxicity against D-17 and A-72 cells. In vivo administration of E134Bf significantly suppressed the development of D-17 and A-72 compared with the control dog IgG in mouse xenografts. These results indicate that E134Bf exerts antitumor effects against dEGFR-expressing canine cancers and could be valuable as part of an antibody treatment regimen for dogs.

Trophoblast cell surface antigen 2 (TROP2) has become one of the effective therapeutic targets by antibody-drug conjugates (ADCs) such as sacituzumab govitecan which US FDA approved in 2020. TROP2 is reported to be overexpressed, and involved in tumor cell proliferation, invasion, metastasis, and poor prognosis in several types of solid tumor. Several clinical trials of anti-TROP2 ADCs are ongoing worldwide in patients with breast and lung cancer. Here, using a Cell-Based Immunization and Screening (CBIS) method, we developed a novel anti-TROP2 monoclonal antibody (clone TrMab-6; mouse IgG_2b, kappa). TrMab-6 was found to be applicable for many experiments, including flow cytometry, Western blotting, and immunohistochemistry. Furthermore, we investigated the potential of TrMab-6 for in vitro antibody-dependent cellular cytotoxicity (ADCC), complement-dependent cytotoxicity (CDC) activities, and in vivo antitumor activities. TrMab-6 strongly induced anti-tumor effects against breast cancer cell lines, including MCF7, MDA-MB-231, and MDA-MB-468 in vitro. In vivo experiments revealed that TrMab-6 significantly reduced tumor growth on breast cancer xenograft models. These results indicated that TrMab-6 could be a promising treatment option for TROP2-expressing breast cancers.

The epidermal growth factor receptor (EGFR) contributes to tumor malignancy via gene amplification and protein overexpression. Previously, we developed an anti-human EGFR (hEGFR) monoclonal antibody, namely EMab-134, which detects hEGFR and dog EGFR (dEGFR) with high sensitivity and specificity. In this study, we produced a defucosylated mouse-dog chimeric anti-EGFR monoclonal antibody, namely E134Bf. In vitro analysis revealed that E134Bf highly exerted antibody-dependent cellular cytotoxicity and complement-dependent cytotoxicity against a canine osteosarcoma cell line (D-17) and a canine fibroblastic cell line (A-72), both of which express endogenous dEGFR. Moreover, in vivo administration of E134Bf significantly suppressed the development of D-17 and A-72 compared with the control dog IgG in mouse xenografts. These results indicate that E134Bf exerts antitumor effects against dEGFR-expressing canine cancers and could be valuable as part of an antibody treatment regimen for dogs.

CD20 is a glycosylated transmembrane protein and is expressed on normal B cells and B cell malignancies. Therapeutic antibodies against CD20 are developed and used in clinic. The understanding of antibody-antigen binding by revealing the epitope is essential for future application to antibody technology. Previously, we developed an anti-human CD20 monoclonal antibody, C20Mab-60 (IgG2a, kappa), using the Cell-Based Immunization and Screening (CBIS). C20Mab-60 can be used for flow cytometry, Western blot, and immunohistochemical analyses. In this study, we examined the critical epitope of C20Mab-60 using enzyme-linked immunosorbent assay (ELISA) with synthesized peptides. We performed ELISA with deletion mutants, and C20Mab-60 reacted to the 160-179 amino acids sequence of CD20. Next, we analyzed the reaction to 20 point mutants, and C20Mab-60 did not recognize the alanine-substituted peptides of N171A, P172A, S173A, and E174A. The results indicate that the binding epitope of C20Mab-60, developed by CBIS, includes Asn171, Pro172, Ser173, and Glu174 of CD20.

Human epidermal growth factor receptor 2 (HER2) is a type I transmembrane 185 kDa protein expressed in various types of normal or cancer cells. Overexpression of HER2 is found in many cancers and is related to cell proliferation, differentiation, and migration. We recently developed a novel anti-HER2 monoclonal antibody, H2Mab-181, by immunizing mice with the purified recombinant extracellular domain of HER2. H2Mab-181 can specifically and sensitively detect HER2 not only in flow cytometry and Western blotting for gastric cancer cell lines, but also in immunohistochemical analyses for gastric cancer tissues. In this study, we analyzed the binding epitope of H2Mab-181 to HER2 using enzyme-linked immunosorbent assay (ELISA). Results showed that the H2Mab-181 epitope was determined to be Gly383, Asp384, Ala386, Asn388, and Pro391 by ELISA.

One of G protein-coupled receptors, CCR9, is mainly expressed in the thymocytes and the small bowel. The ligand of CCR9 is CCL25 (TECK), and the CCR9-CCL25 axis controls T cell maturation and intestinal immune response. CCR9 is related to graft-versus-host disease and autoimmune diseases. Recent studies have been reported that CCR9 is also associated with tumor proliferation, apoptosis, migration, and drug resistance. Therefore, CCR9-targeting therapy is receiving a lot of attention. Previously, we developed an anti-human CCR9 (hCCR9) monoclonal antibody, C9Mab-1 (IgG1, kappa), which can be used for flow cytometry, by immunizing mice with hCCR9-overexpressed Chinese hamster ovary-K1 cells. In this study, we examined the critical epitope of C9Mab-1, using enzyme-linked immunosorbent assay (ELISA) with synthesized peptides. First, we performed ELISA with deletion mutants, and C9Mab-1 reacted to the 1-20 amino acids sequence of hCCR9. Next, we analyzed the reaction to 20 point mutants, and C9Mab-1 did not recognize the alanine-substituted peptides of I10A, P11A, N12A, M13A, A14G, D16A, and Y17A. The results indicate that the binding epitope of C9Mab-1 includes Ile10, Pro11, Asn12, Met13, Ala14, Asp16, and Tyr17 of hCCR9.

CD20 is one of the B-lymphocyte antigens and an effective target for the detection and treatment of B cell lymphomas; specific and sensitive monoclonal antibodies (mAbs) are required thus for their diagnosis. Recently, we developed a novel anti-CD20 mAb (clone C20Mab-60), which is not only useful for flow cytometry but also for Western blot and immunohistochemical analyses. However, the epitope of C20Mab-60 has not been determined. To clarify the binding region of mAbs against their target molecules, it is essential to understand the pharmacological function of each mAb. In this study, we aimed to identify the epitope of C20Mab-60 for CD20 using the novel histidine tag (His-tag) insertion for epitope mapping (HisMAP) method. We first established an anti-His-tag mAb, HisMab-1 (mouse IgG2b, kappa), by immunizing mice with recombinant proteins containing an N-terminal His-tag. Although HisMab-1 detected the 4x, 5x, and 6xHis tag-inserted CD20 proteins using flow cytometry, 5xHis tag was selected. While HisMab-1 recognized all the 5xHis tag-inserted CD20 from the 142nd to the 183rd amino acid (aa), C20Mab-60 did not react with the 5xHis tag-inserted CD20 from the 171st to the 174th aa. These results indicate that the main epitope of C20Mab-60 for CD20 is a peptide from 171st to 174th aa of CD20. HisMAP method could be advantageous in the determination of the critical epitope of functional mAbs against many target molecules.

Podocalyxin (PODXL) is a type I transmembrane sialoglycoprotein that is overexpressed in human cancers, including breast, oral, and lung. PODXL promotes tumor progression, and its expression is associated with poor prognosis. Since PODXL is expressed in normal cells, including kidney podocytes and vascular endothelial cells (VECs), cancer-specific monoclonal antibodies (mAbs) are necessary to reduce the adverse effects of antibody therapy on PODXL-expressing cancers. Previously, we established a cancer-specific mAb against PODXL, PcMab-60 (mouse IgM, kappa), by immunizing mice with soluble PODXL produced by LN229 glioblastoma cells. PcMab-60 reacted with PODXL-expressing cancer cells, but did not react with VECs. In this study, we investigated an epitope of PcMab-60 using flow cytometry, surface plasmon resonance (SPR), and enzyme-linked immunosorbent assay (ELISA). The results of SPR revealed that the PcMab-60 epitope consisted of Thr105, Arg109, Gly110, Gly111, Gly112, Ser113, Gly114, Asn115, Pro116, and Thr117. In contrast, the results of ELISA revealed that the PcMab-60 epitope consisted of Arg109, Gly110, Gly111, Gly112, Ser113, Gly114, Asn115, and Pro116. These results demonstrate the cancer-specific epitope, which was recognized by PcMab-60.

CD44 is a type I transmembrane protein expressed in various kinds of normal cancer cells, including pancreatic, breast, and oral cancers. CD44 is associated with cancer progression, metastases, and treatment resistance. CD44 consists of 20 exons, and various isoforms exist due to alternative splicing of the central 10 exons. Some splicing variants show cancer-specific expression patterns and are related to prognosis of patients with cancer. Therefore, CD44 targeting therapy has been attracting attention. In a previous study, we established an anti-CD44 monoclonal antibody, C44Mab-46 (IgG1, kappa), useful for flow cytometry, Western blotting, and immunohistochemistry by immunizing mice with CD44v3-10 ectodomain. This study investigated the binding epitope of C44Mab-46 using enzyme-linked immunosorbent assay (ELISA) and the surface plasmon resonance (SPR) with the synthesized peptide. ELISA results using deletion mutants showed that C44Mab-46 reacted with the amino acids (aa) of 161-180 aa of CD44. Further examination of the C44Mab-46 epitope using ELISA with point mutants in 161-180 aa of CD44 demonstrates that the C44Mab-46 epitope comprised Thr174, Asp177, and Val178. The SPR with point mutants in 161-180 aa of CD44 demonstrated that the C44Mab-46 epitope comprises Thr174, Asp175, Asp176, Asp177, and Val178. Together, the C44Mab-46 epitope was determined to be located in exon 5 of CD44.

Induction of cellular senescence in cancerous cells is an important strategy which is used in the treatment of cancer. However, cancer cells are capable of exhibiting resistance to cellular senescence through inactivation of tumor suppressors. Because of this, establishment of a route to cellular senescence induction in cancer cells is a crucial direction for developing future cancer therapies. In this study, we demonstrate the involvement of TSC-22 homologous gene-1 (THG-1, also called TSC22D4) in the suppression of cellular senescence. CRISPR/Cas9 gene editing was used to establish THG-1 knockout (KO) cells in a THG-1 positive esophageal tumor cell line. It was found that THG-1 KO cells exhibited delayed cell proliferation as well as cellular senescence. The elevated expression of the CDK inhibitor P21(CDKN1A) was also identified in senescent cells. Through the investigation of the upstream pathway for induction of P21(CDKN1A), the JUNB pathway was identified to play a critical role in P21(CDKN1A) transcription; in fact, the siRNA-mediated knockdown of JUNB reduced the abundance of P21(CDKN1A) mRNA and cellular senescence in THG-1 KO cells. These findings provide a novel insight

Knockdown of THG-1 in TE13 esophageal squamous cell carcinoma (ESCC) cells is known to suppress tumorsphere growth. THG-1 was identified as an NRBP1 binding protein, and NRBP1 was reported to downregulate an stemness-related transcriptional factor SALL4, so we decided to examine the possibility that tumorigenic function of THG-1 is achieved by the competition to the tumor-suppressive function of NRBP1. SALL4 was decreased in THG-1 deficient TE13 cells with reduced tumorsphere formation, while exogenous SALL4 expression in THG-1 deficient TE13 cells recovered expression of stemness genes (NANOG and OCT4) and partially, but significantly, recovered tumorsphere formation ability. Additionally, we found that NRBP1 induced ubiquitination of SALL4, and THG-1 interrupted the ubiquitination of SALL4 by antagonizing NRBP1 binding to SALL4. These results suggest that THG-1 promotes tumorsphere growth of ESCC cells by the stabilization of SALL4 protein and induction of the target stemness genes through competitive binding to NRBP1.

Fungal secondary metabolites, (+)-WIN 64821 and (-)-ditryptophenaline, are biosynthesized through condensation of L-tryptophan and L-phenylalanine followed by reductive dimerization with generation of stereochemical variations. Inspired by the stereo-divergent biogenetic process, a collection of bispyrrolidinoindoline diketopiperazine alkaloids and their analogs were designed and synthesized with systematic diversification of the stereochemistry of the privileged structural motif of the fungal alkaloids. Not only the stereochemical modifications of (+)-WIN 64821 at the 3, 11, and 15 positions, but also ring cleavage of the diketopiperazine moieties allowed generation of a lead compound exhibiting potent growth inhibitory activity (IC50 = 3.03 µM) toward human colon cancer cells. Structure-activity relationship studies revealed that all three stereogenic centers were essential for the pharmacophore. High cell densities dramatically intensified the cytotoxic activities of the lead compound.

Glycoprotein nmb (GPNMB) is a type I transmembrane protein that contributes to the initiation and malignant progression of breast cancer through induction of epithelial-mesenchymal transition (EMT). Although it is known that EMT is associated with not only cancer invasion but also acquisition of cancer stem cell (CSC) properties, the function of GPNMB in this acquisition of CSC properties has yet to be elucidated. To address this issue, we utilized a three-dimensional (3D) sphere culture method to examine the correlation between GPNMB and CSC properties in breast cancer cells. 3D sphere cultures induced higher expression of CSC genes and EMT-inducing transcription factor (EMT-TF) genes than did the 2D monolayer cultures. 3D culture also induced cell surface expression of GPNMB on limited numbers of cells in the spheres, whereas the 2D cultures did not. Therefore, we isolated cell surface-GPNMBhigh and -GPNMBlow cells from the spheres. Cell surface-GPNMBhigh cells expressed high levels of CSC genes and EMT-TF genes, had significantly higher sphere-forming frequencies than the cell surface-GPNMBlow cells, and showed no detectable levels of proliferation marker genes. Similar results

The alpha2(I) collagen gene shows cell type-specific expression, however, the mechanism behind this specificity remains to be determined. We demonstrate here that transforming growth factor-beta (TGF-beta)-mediated induction of alpha2(I) collagen gene is regulated by DNA methylation in a cell type-specific manner. Human alpha2(I) collagen mRNA and type I collagen protein were expressed in normal human fibroblasts (NHF), and also strongly enhanced by TGF-beta; they were not detected in HaCaT, HeLa, or HepG2 cells (termed "collagen-induction resistant (CIR) cells") even following stimulation with TGF-beta. On the other hand, the transcriptional activity of exogenously transfected alpha2(I) collagen promoter was clearly up-regulated by TGF-beta in the CIR cells as well as in NHF. In the CIR cells, CpG clusters around the transcription start site of the alpha2(I) collagen gene were heavily methylated, whereas no methylation was detected in NHF. Moreover, alpha2(I) collagen gene was reactivated in the CIR cells by 5-Aza-2-deoxycytidine (5-AdC) treatment to some extent. However, demethylation by 5-AdC was limited and it was unable to recover the TGF-beta responsiveness. In NHF, the alpha2(I) collagen gene has a Smad3-accessible chromatin structure and acetylated histones in the promoter regions. By contrast, in the CIR cells, Smad3 failed to bind to the chromatin and histones were not acetylated in this area. Furthermore, in vitro methylation of the reporter gene containing the alpha2(I) collagen promoter significantly reduced both basal and TGF-beta-induced enhancement of the transcriptional activity in NHF. Thus, we propose that alpha2(I) collagen gene provides the first example of the TGF-beta responsive gene whose cell type-specificity is regulated by CpG methylation.